Monolayer Maintenance of iPSC

Creative Biolabs is a leading service provider that focuses on iPSC generation. With years of experience, now we can provide the iPSC maintenance service for our customers all over the world.

Introduction of iPSC Maintenance

Due to the similar properties compared with embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) enable the next generation of cell therapies in clinical applications. Since iPSCs were generated from mouse embryonic fibroblasts and adult human somatic cells by introducing four defined reprogramming factors in 2006, several reprogramming strategies have been discovered for the iPSC generation. However, there are still several technical hurdles before a wide range of clinical implementation, such as the scalable, along with reproducible production of iPSCs. With years of experiment, scientists at Creative Biolabs have built two systems for iPSCs culture, which are feeder-dependent culture system and feeder-free culture system.

Fig.1 Flow chart for general maintenance and passaging of iPS cells grown on MEFs.

Feeder-dependent Culture System

iPSCs are derived from adult cell types such as fibroblasts using various strategies. The common feeder-dependent culture system provided by Creative Biolabs is based on iPSCs maintained on a layer of inactive murine embryonic fibroblast (MEF) feeder cells. Under these conditions, the human iPSCs can be maintained for several passages without compromising cells' proliferation or pluripotency and differentiation potential. This feeder-dependent culture system can benefit from its high efficiency in a short time but sometimes limited by the requirement of significant time and effort.

Brief Steps:

• MEF dishes preparation
• Human iPSCs thawing and plating
• Human iPSCs passaging

Feeder-free Culture System

In order to discover a novel culture system applicable for human iPSCs, both feeder-free and xeno-free conditions must be met. There are various matrices that can be used to replace feeder cells, such as CELLstart, synthetic polymers, matrigel, and recombinant proteins. Meanwhile, the available xeno-free media includes TeSR2, NutriStem, and Essential E8 medium. The next step is to identify an efficacious combination of matrix and medium, since medium require configuration to satisfy cells in new condition with potential problems such as detachment, poor metabolism, difficulty in proliferation and possible apoptosis. After a series of experiment, we have successfully developed the novel feeder-free culture system for human iPSCs using StemFit™ and rLN511E8. With the advantages of simple use, high expand, and reproduction, the human iPSCs with equivalent growth and differentiation potential can be generated from various tissues under our feeder-free and xeno-free conditions.

Fig.2 Feeder-free culture method for human iPSCs.

With our well-established iPSC generation technology, Creative Biolabs now provides different culture systems for iPSC generation. Based on your specific situation, we can also customize protocol to meet our clients' demands precisely. Creative Biolabs provides other services regarding iPSC generation and iPSC applications, please do not hesitate to contact us if you are interested in them.

Services at Creative Biolabs

Our services focus on the cultivation, expansion, and upkeep of cells grown in a monolayer. This service contributes to the critical field of stem cell research and provides researchers with the quality materials they need.

Here's how our process would potentially look:

• Cell Cultivation: With specialized expertise in stem cell biology, we provide cultivation services to maintain the stem cell's pluripotency and stability. Our professionals use a high-affinity matrix solution in cell culture dishes to create a suitable environment for the cultivation of iPSC.
• Routine Maintenance: We regularly feed and sub-culture cells to ensure they grow and divide correctly. We strictly follow cell handling protocols to mitigate contamination risk and maintain the stem cells' integrity.
• Growth Monitoring: By closely observing the monolayer under a microscope, we keep a detailed record of growth, shape, and morphology of iPSCs. This is crucial to assessing the viability and health of stem cells.
• Expansion: Once the stem cells reach the desired confluency (typically around 70%-80%), we perform cell passaging, a process to sustain their exponential growth and to prevent overcrowding within the culture dish.
• Preservation & Cryopreservation: We also offer preservation services, ensuring the long-term maintenance of your iPSC lines by cryopreserving them, providing storage under optimal conditions in liquid nitrogen.
• Quality Control: Each iPSC line undergoes rigorous quality control including tests for sterility, cell morphology, growth characteristics, pluripotency, and absence of mycoplasma contamination.
• Data Reporting: We offer comprehensive progress reports to keep you updated on the status of your iPSC.

Our monolayer maintenance services of iPSC ensure optimal cell health and are performed under stringent safety standards. They are invaluable for studies related to cellular aging, genetic disorders, drug discovery, and more.

Features of Our Services

• Versatility - Our service is geared towards the support of various iPSC lines, making it versatile and a perfect match for diverse experimental outlines and designs.
• Time and Cost Saving - Removal of the need to train and maintain skilled staff in-house for monolayer maintenance, or to invest in the necessary specialty equipment, represents significant financial and time savings.
• Novel Techniques - We use cutting-edge techniques and protocols to ensure minimal variation and maximal cell viability. Our team stays updated with the latest trends and advancements in iPSC handling to provide the finest service to our clients.
• Full Documentation - We provide complete and detailed records for each batch, including cell counts, viability, phenotype, and sterility. This is incredibly helpful in maintaining transparency and traceability of your research.
• Customer Support - Our team presents comprehensive support services including free consultations, tailor-made experimental design, troubleshooting, and prompt response to any queries or requests.

FAQs

• Q: What measures do you take to optimize cell viability and minimize cell stress during the maintenance process?
  A: We prioritize the well-being of your iPSCs throughout the maintenance process. Our optimized culture conditions, including appropriate media formulations, substrate coatings, and controlled incubation environments, are designed to promote cell viability and minimize stress. Regular assessment and adjustments ensure that cells remain healthy and robust.
• Q: How does this service contribute to scientific experiments?
  A: The quality of iPSC is fundamental to the success of scientific experiments. The service ensures that these crucial cells are maintained in optimal conditions, enhances their capability to differentiate into various types, and thus contributes profoundly to research success notably in disease modeling, drug discovery and development, and potential therapeutic applications.
• Q: Is this service suitable for long-term projects?
  A: Yes, our services are perfectly tailored for both short-term and long-term projects. We monitor and adjust the care of the iPSCs depending on the duration and requirements of the project, ensuring they retain their pluripotency and health throughout.
• Q: What steps are taken if iPSCs begin to differentiate prematurely?
  A: If we detect early signs of differentiation, we would immediately separate the differentiated cells from the population to maintain the undifferentiated state of the remaining cells. This is done either by manual dissection under a microscope or by enzymatic methods.
• Q: Beyond maintenance, do you offer any other related services for iPSCs?
  A: Absolutely. Apart from maintenance services, we also offer services like iPSC generation, characterization, cryopreservation, resuscitation, and differentiation. Our team is skilled and experienced in handling all aspects related to iPSC research.

Scientific Resources

iPSC Culture

Culture Protocol

Learn More

References

1. Sugii, S. (2011). “Feeder-dependent and feeder-independent ips cell derivation from human and mouse adipose stem cells.” Nature Protocols 6(3), 346-58.
2. Nakagawa, M. (2014). “A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells.” Scientific Reports 4(1), 3594.

For Research Use Only. Not For Clinical Use.