Human induced pluripotent stem cells (iPSCs) represent one of the most versatile tools in modern biomedical research. Capable of differentiating towards virtually any cell fate, they serve as a powerful platform for disease modeling, drug discovery, and regenerative medicine applications. Preserving this utility, however, depends critically on maintaining a consistently undifferentiated state throughout the full duration of culture.
iPSC culture is inherently demanding. Routine maintenance involves a tightly coordinated series of steps, including extracellular matrix (ECM) coating, cell seeding, media exchanges at regular intervals, confluency monitoring, and timely passaging.
Each of these steps must be executed with precision to prevent spontaneous differentiation and preserve pluripotency. Parameters such as detachment reagent selection, incubation timing, pipetting technique, and seeding density each influence culture outcome, and small deviations in any one of them can trigger phenotypic drift that compromises experimental consistency and data quality. Sustaining this level of control manually, across multiple iPSC lines and plate formats, simultaneously is labor-intensive and difficult to scale.
Cellmatic®: Integrated Automation for the Full iPSC Culture Workflow
The Cellmatic automated cell culture system (CCS) consolidates the entire iPSC maintenance workflow into a single, walk-away platform. ECM coating, cell plating, media exchange, confluency monitoring, and passaging are all executed automatically and on schedule, under unified software control through the Suite application. By removing manual intervention from routine culture steps, the Cellmatic eliminates operator-dependent variability and enables consistent, reproducible iPSC maintenance across multiple lines and plate formats in parallel.
Inside the Cellmatic, liquid handling steps are performed by two complementary modules, the FLO i8® PD and the Reagent Exchanger (RX), while culture monitoring is carried out by the integrated Imager by the Formulatrix. Plate transfers between modules are handled autonomously by the Stack® autonomous plate handler.
iPSC common research workflow
Automated ECM-Coating of iPSC Culture Plates inside the Cellmatic
The Cellmatic platform supports automation of the iPSC culture workflow, with a current focus on two-dimensional (2D), feeder-free systems. In feeder-free culture, the mitotically inactivated fibroblast feeder layer that normally supplies growth factors and extracellular matrix (ECM) support is replaced by a thin ECM gel coating on the culture surface. This coating typically contains matrix proteins such as collagens, laminins, and fibronectins, alongside proteoglycans and growth factors, and functions as a basement membrane that enables iPSC attachment after seeding. This ECM layer is critical for maintaining the quality of 2D iPSC cultures, as cell–matrix adhesion supports the cell–cell interactions required for sustained proliferation and pluripotency. In contrast, direct contact with the uncoated plastic surface promotes early differentiation, marked by altered morphology and cell spreading. Consequently, consistent preparation of ECM-coated daughter plates is essential for feeder-free iPSC maintenance and downstream experimentation.
Feeder-free 2D iPSC monolayer culture on a coated surface
[Source: BCH stem cell core lab]
Within the Cellmatic, ECM coating is fully automated. The liquid handler dispenses diluted ECM solution from registered troughs, with support for a wide range of commercially available matrix products. When using Matrigel®, for example, the Suite application can automate daughter plate preparation by scheduling incubation at 37°C, with plates automatically transferred to the integrated incubator via Stack, and subsequently schedules the post-gelation media exchange to align with upcoming passaging workflows, thereby ensuring that ECM-coated plates are ready precisely when needed, without manual coordination. ECM plating protocols can also be run in batch, with coated plates stored in the Cellmatic Cold Store until needed for seeding.
Automated iPSC Passaging inside the Cellmatic
iPSCs grow in tightly packed colonies, where strong cell–cell adhesion maintains the undifferentiated state, typically reflected by a high nucleus-to-cytoplasm ratio. As in other 2D cultures, iPSC monolayers are ready for passaging at approximately 70–85% confluency. Routine maintenance is usually performed by passaging cells as clumps or aggregates to preserve long-term pluripotency, whereas single cell passaging can be used for specific applications such as differentiation or reprogramming. In both cases, successful passaging depends on careful control of detachment reagent type, incubation conditions, and pipetting technique.
Plate being passaged
Cell dislodging, mixing and seeding into daughter plates
Uniform clump size (50–200 µm) depends on careful control of detachment incubation time and post-detachment mixing to prevent over-dissociation. Within the Cellmatic, these parameters are fully adjustable, with multiple mixing approaches available, including the FLO i8 PD tip mixing, plate rocking, and the Formulatrix Dislodger plate shaker. This flexibility accommodates both clump-based and single-cell passaging protocols, allowing each to be optimized for the desired outcome and cell line. Excessive trituration should be avoided to minimize shear stress on the cells. The resulting cell suspension can be seeded using either split ratio or cell count, and the culture media is typically supplemented with Rho-associated kinase (ROCK) inhibitor, Y-27632, immediately following dissociation to mitigate mechanical stress-induced cell death and improve cell survival during the initial attachment phase.
The growth of newly plated iPSCs can be monitored using the Formulatrix Imager, which enables automated confluency assessment at user-defined time intervals. This functionality supports the implementation of confluence-based automated workflows, such as triggering passaging upon reaching a predefined threshold (e.g., 80% confluence). Continuous imaging also facilitates early detection and documentation of culture abnormalities, including unintended differentiation.
iPSC culture maintenance and experimental workflow
Automated Media Exchange and Support for Differentiation Workflows
Routine media exchange within the Cellmatic is scheduled through the Suite application and executed automatically by the FLO i8 PD or RX. In standard iPSC maintenance, media exchange is initiated 24 hours post-seeding and subsequently performed every one to two days, replenishing growth factors consumed by proliferating cells, removing residual ROCK inhibitor, and clearing detached cells or debris. Both full and partial media exchange are supported, with optional wash steps prior to fresh media addition.
Beyond routine iPSC maintenance, flexible media exchange is critical for applications such as in vitro differentiation and iPSC reprogramming. Both processes depend on multiple, precisely timed media exchanges. In many protocols, culture medium is replaced gradually and in partial volume at each step, minimizing cellular stress while supporting progression towards the desired cell fate. The Cellmatic supports these demanding workflows through customizable media exchange modes, including full and partial volume replacement with user-defined scheduling. This enables scheduled media exchanges required for differentiation and reprogramming protocols, and can be further tailored to meet the specific timing and volume requirements of individual experimental designs.
iPSC maintenance within the Cellmatic platform can be performed in various multiwell formats including 1-well to 384-well plates in both circular and square well configurations, enabling parallel processing of multiple iPSC lines within a single plate. The use of non-contact dispensing via RX nozzles and FLO tips minimizes the risk of cross-contamination between wells, which is particularly advantageous for clonal expansion assays in 96- or 384-well plate formats. Moreover, this flexible multiwell plate handling supports scalable expansion across different source and target plate formats, such as passaging from a 96-well plate to a 6-well plate, or from a 6-well plate to a single-well format.
Media exchange in a 1-well plate with nozzle cleaning
Media exchange in a 96-well plate
Webinar
Automating hiPSC Workflows with the Formulatrix Cellmatic™
hiPSC cultures are prone to variability, making scalable, standardized automation essential for reproducible results. In this webinar, Dr. Thorsten Schlaeger from Boston Children's Hospital demonstrates how the Cellmatic automates hiPSC expansion, media exchange, passaging, and confluency monitoring to improve throughput and consistency.
Application Notes
iPSC Maintenance in a 6-well Plate Using the Cellmatic™
iPSC maintenance demands daily monitoring, media exchange, and passaging all labor-intensive and prone to variability. The Cellmatic automates these tasks to deliver consistent, reproducible iPSC cultures with reduced manual effort.
Automated Gentle Media Exchange for Loosely Adherent HEK-293T Cells with the Reagent Exchanger (RX)
HEK-293T cells are prone to detachment, making gentle liquid handling critical during media exchange. The RX automates aspiration and dispensing, reducing cell loss compared with manual pipetting.
