As a cutting-edge technology in the stem cell field, induced pluripotent stem cells (iPSCs) have demonstrated significant technical barriers and broad market potential, thanks to their unlimited proliferative and differentiative capacity, coupled with breakthrough reprogramming technologies. Projections indicate that the global stem cell market will reach $270.5 billion by 2025, with the iPSC segment boasting a compound annual growth rate (CAGR) exceeding 13.8%.
Behind this industrial advancement lie the unique biological properties of iPSCs: they can self-renew to maintain a stable cell population and differentiate into nearly all cell types in the human body, including neural cells, cardiomyocytes, and pancreatic cells. Derived from somatic cells, iPSCs eliminate restrictions on sample collection, making them an ideal "seed" source for cell therapies targeting various diseases.
However, iPSCs are extremely sensitive to even minor changes in the culture environment, often leading to challenges such as unwanted differentiation and poor adherence. Ensuring the maintenance of pluripotency and prevention of spontaneous differentiation requires precise control of the culture conditions. This article synthesizes the core elements of iPSC culture, systematically outlining key processes, causes of differentiation, and corresponding solutions to serve as a reference for standardized cultivation.
I. Establishing the Culture System
The maintenance of iPSC stemness relies on precise microenvironment regulation, focusing on three core components: culture medium, substrate, and environmental conditions.
1. Culture Medium: Balancing Nutrition and Signaling
As the "nutritional source" for iPSC growth, the medium formula directly impacts cell status, playing a pivotal role in maintaining stability and functionality. Serum-free media (e.g., Yocon’s iPSC Serum-Free Medium Kit) are recommended as the preferred choice. These media contain optimized growth factor combinations that activate stemness-maintaining pathways such as FGF2/ERK, supporting feeder-free culture, effectively inhibiting spontaneous differentiation, and enabling long-term passaging.
A critical operation: FGF-2 has a half-life of less than 24 hours, requiring timely supplementation to avoid differentiation due to insufficient signaling. Yocon’s iPSC Serum-Free Medium uses thermostable FGF-2, which offers a longer half-life and higher, more stable biological activity, supporting medium changes every other day.
2. Substrate: Adhesion and Growth Support
Early iPSC culture often relied on mouse embryonic fibroblasts (MEFs) as feeders, which secrete factors that promote iPSC growth and pluripotency maintenance. However, MEFs carry the risk of xenogenic contamination, making feeder-free culture the current mainstream. Feeder-free systems commonly use matrix gels such as Matrigel—a basement membrane extract that mimics the extracellular matrix, providing optimal adhesion and growth support for iPSCs.
A critical operation: When using Matrigel, aliquot according to the manufacturer’s recommended volume based on the batch number. Dilute each aliquot with 36mL of cold DMEM/F12, coat the wells, and perform the entire process on ice. Incubate at 37°C for 1-2 hours to ensure full matrix polymerization, creating a stable adhesive surface for cells.
3. Environment: Precise and Stable Control
A stable and suitable gas environment and temperature are essential for healthy iPSC growth. Incubators should be set to 37°C to maintain normal cell metabolism and physiological activities, with 5% CO₂ to stabilize the medium’s pH. Additionally, monitor incubator humidity to prevent medium evaporation—place a water tray at the bottom and replenish water regularly.
II. Core Operational Processes and Key Details
1. Passaging
iPSCs grow in colonies and are ready for passaging when colonies become large, with dense, bright centers (compared to their edges), and adjacent colonies start to fuse. The passaging cycle ranges from 3 to 5 days, depending on the size and density of the inoculated cell clusters. Passaging too early or frequently can lead to poor adhesion, reduced yield, and differentiation. Conversely, delayed passaging results in overcrowding, nutrient competition, and differentiation (e.g., altered cell morphology, decreased expression of pluripotency markers), compromising experimental results.
Gentle digestive enzymes containing EDTA (e.g., Accutase or Tryple) are commonly used for dissociation. EDTA chelates extracellular calcium, weakening cell-cell and cell-matrix adhesion to enhance digestion efficiency. Control digestion time to 3-5 minutes to avoid over-digestion.
After terminating digestion, pipette gently and passage as single cells or small clusters. iPSCs have poor viability and stemness maintenance in the single-cell state, making spontaneous differentiation more likely. If single-cell passaging is required, add Y27632 to a final concentration of 10μM.
Passage when cell confluency reaches approximately 90%, with a recommended ratio of 1:20 (adjustable based on actual confluency). Count cells before passaging—seed 100,000-200,000 cells per well in a 6-well plate to ensure sufficient growth space while maintaining optimal cell density for intercellular signaling and growth.
2. Cryopreservation
Before cryopreservation, carefully remove differentiated regions under a microscope using a pipette tip or other precision tools. Differentiated cells deviate from stem cell characteristics and may disrupt the stability of the entire culture system after thawing. Ensure only differentiated cells are removed without damaging healthy iPSCs. Cryopreserve iPSCs when they are in good condition and at appropriate density.
3. Quality Control
(1) Genomic Stability Monitoring
Long-term iPSC culture may induce gene mutations, affecting cell quality and application safety. Conduct karyotype analysis regularly (e.g., every 10-20 passages) using chromosome G-banding to check for abnormalities in chromosome number, morphology, and structure. Promptly eliminate abnormal cells to prevent their accumulation in the culture system.
(2) Pluripotency Identification
Pluripotency is the core characteristic of iPSCs, identifiable by detecting the expression of pluripotency markers such as TRA-1-60 and Oct4. Methods include immunofluorescence staining (visualizing marker localization and intensity), flow cytometry (quantifying positively expressing cells), and RT-PCR (detecting marker expression at the mRNA level). Only iPSCs with high expression of pluripotency markers possess strong application potential.
III. Common iPSC Culture Issues and Solutions
1. Differentiation: Root Causes and Effective Remedies
(1) Core Causes
Microenvironment imbalance: Batch-to-batch variations in matrix gels alter adhesion and growth factor signals, disrupting stemness maintenance. Mechanical damage from excessive pipetting also triggers cellular stress responses and differentiation.
Metabolic stress: iPSCs have high metabolic activity, producing lactate and other metabolites. Delayed medium changes lead to lactate accumulation and decreased pH (below 7.2), interfering with enzyme activity and signaling pathways, and promoting differentiation. Insufficient supplementation of short-half-life growth factors (e.g., FGF-2) weakens stemness-maintaining signals.
Cell overcrowding: iPSCs lack contact inhibition. Overgrown colonies cause nutrient depletion and waste accumulation in central cells, inducing ectodermal differentiation. Imbalanced contact inhibition in edge cells promotes mesodermal differentiation.
(2) Remedies
High-ratio passaging (≥1:20): Dilute differentiated cells and select high-quality colonies (dense morphology, clear edges, good refractivity) to maintain pluripotency.
Local treatment for low differentiation rates: Carefully scrape differentiated regions under a microscope with a 10μL pipette tip, avoiding damage to undifferentiated cells and the matrix layer. Replace with fresh medium immediately to eliminate residual differentiation-inducing signals.
Inhibitor addition for trilineage differentiation trends: Add appropriate concentrations of inhibitors and monitor cell status closely. Gradually reduce inhibitor dosage after 3-5 passages to avoid adverse effects on stemness.
Discard severely differentiated cultures: If differentiated regions occupy most of the colonies, discard the batch and thaw a new vial.
2. Low Thawing Efficiency: Success Depends on Operational Details
(1) Common Causes
Excessive pipetting disperses cells into single cells, which have poor viability and stemness maintenance. Undersized cell clusters after thawing also reduce survival due to disrupted intercellular support.
(2) Optimization Measures
Adjust pipetting technique: Pipette gently and minimize the number of strokes to reduce mechanical damage.
Check cluster size post-thaw: Use a microscope to verify optimal cluster size. Adjust pipetting 力度 and frequency in subsequent thawing to form larger, more stable clusters.
Add Y27632 during thawing to improve cell survival.
3. Poor Adhesion After Passaging: Digestion and Cluster Size Are Key
(1) Common Causes
Over-digestion: Prolonged digestion degrades cell surface adhesion molecules, impairing adherence.
Improper pipetting: Excessive or forceful pipetting reduces cluster size, decreasing contact area with the culture dish and causing mechanical damage.
Under-digestion: Insufficient digestion requires forceful pipetting to detach cells, damaging cell membranes and cytoskeletons.
Incomplete matrix coating: Premature matrix gel solidification (due to improper handling) leads to uneven coating.
(2) Optimization Measures
Control digestion time: Monitor cells under a microscope; terminate digestion immediately when cells slightly round up and membranes wrinkle.
Improve pipetting technique: Reduce strokes and maintain uniform, moderate cluster size.
Extend digestion time slightly for stubbornly attached cells to minimize mechanical interference.
Perform matrix coating on ice to prevent premature solidification.
IV. Yocon’s iPSC Serum-Free Medium Kit
With years of focus on R&D and production of serum-free culture products for cell therapy, Yocon has launched the iPSC Serum-Free Medium Kit. Specifically designed for the stable expansion and long-term maintenance of human induced pluripotent stem cells (hiPSCs), this kit offers high efficiency, stability, and defined composition, suitable for diverse applications.
Key features of the iPSC Serum-Free Medium Kit:
Chemically defined serum-free medium for in vitro expansion and long-term culture of hiPSCs.
Scientifically optimized formula with thermostable cytokines and a robust buffering system.
Supports medium changes every other day and single-cell passaging, enabling simple and versatile operation.
Superior expansion performance: Supports over 30 passages in feeder-free culture, with normal karyotype and no significant differentiation.
iPSCs continue to revolutionize regenerative medicine and drug development. By mastering the core principles of culture system establishment, standardized operations, and troubleshooting, researchers can ensure the quality and stability of iPSCs, unlocking their full potential in clinical translation and scientific research.
