You never forget the first time you thaw a vial of induced pluripotent stem cells. Standing in the lab, holding a precious vial of cells, reading, and re-reading the protocols, and crossing your fingers that you don’t kill the cells, you gingerly hold that vial in a waterbath like it is a rare diamond. In almost 20 years of cell culture experience, no cell type has inspired as much fear and awe in my scientific heart than that vial of iPSCs.
Figure 1. The number of publications referencing the term “iPSC” in PubMed since 2006.
Since their discovery in 2006 by Takahashi and Yamanaka, induced pluripotent stem cells (iPSCs) have seen a meteoric rise in their use throughout academic and industrial research, and to date, there are over 21,000 publications in PubMed referencing them (Figure 1). Generated by reprogramming terminally differentiated somatic cells with four key transcription factors (Oct4, Sox2, KLF4, and c-myc), human iPSCs have the potential to unlimitedly proliferate and differentiate into all of the key cell types in the human body. These cells hold promise for a myriad of therapeutic research areas, including gene therapy, regenerative medicine, and personalized medicine. In addition, they alleviate many of the ethical concerns and sourcing issues associated with pluripotent cells such as human embryonic stem cells. And yet, despite countless research, rapidly advancing protocols, and increased access for researchers at all levels, iPSCs remain a challenging enigma that pose some key limitations for their utility in therapeutic research.
Common Challenges with iPSCs
So, what is it about iPSCs that can strike fear into the heart of an experienced bench scientist?
- The process of generating a reprogrammed iPSC line from a somatic cell is inefficient, highly manual, and labor intensive, with a success rate of less than 1%.
- Even established iPSC lines are sensitive and easily perturbed, requiring constant maintenance and attention to viability, morphology, and confluence.
- Poor culture conditions and cell line instability can lead to spontaneous differentiation in culture and a loss of pluripotency.
- Generating edited cell lines via CRISPR or other technologies is incredibly difficult, with low efficiency and lack of monoclonality.
- The labor, cost, and reagent burden to maintain iPSC workflows is incredibly high and often prohibitive.
To overcome these bottlenecks and challenges to iPSC growth, there are a variety of specialized medias, additives, and tissue culture dish coatings that are meant to improve cell survival in vitro. However, despite these options, cell viability is often compromised, and the phenotype of the line can become unstable with prolonged culture. In addition, iPSC lines require constant maintenance, necessitating daily media changes and manual manipulation to remove areas of differentiation or to isolate desired clones for further study.
A New Approach
The improvement of iPSC culture conditions and the development of automated iPSC workflows have the potential to increase the utility, ease, and throughput of these workflows, thereby accelerating the use of iPSCs in personalized medicine and drug discovery.
A novel solution that enables this possibility is the CellRaft® Technology from Cell Microsystems. Briefly, the key features of the technology that are beneficial for iPSC workflows include:
- Improved viability of single-cell iPSCs due to flask-like culture conditions in the CellRaft Array
- Track and trace capability of single cells to clone using software-guided cell selection
- Clonality and pluripotency assurance via on-platform analysis
- Fully automated workflow that decreases cost and time per clone, while screening 500X more cells per consumable than a standard 96 well plate.
Case Studies
To determine whether these key features could alleviate bottlenecks hindering iPSC workflows, we compared clonal iPSC development using CellRaft Technology to traditional limiting dilution. Using this method, we were able to generate over 200 single cell derived iPSC clones on a single consumable, compared to 10 clones in a single 96 well plate (Figure 2). This workflow required 1000X less iPSC coating and 2000X less media per cell screened. In addition, the image acquisition over time allows for the detailed assessment of monoclonality, ensuring that successful clones are not heterogeneous and eliminating the need for downstream clonal characterization and shortening the time to use in downstream applications (Figure 3).
Figure 2: CellRaft AIR vs limiting dilution for monoclonal iPSC development. iPSCs were seeded on either a CellRaft Array coated with iMatrix-511 (Matrixome) or h-ESC Matrigel (Corning) or on iMatrix-511 coated 96 well plates for limiting dilution. Colony formation was monitored using the CellRaft AIR or manual observation.
Figure 3: Track and trace of iPSC clone formation on the CellRaft AIR. Four different iPSC cell lines were seeded on CellRaft arrays on one of three coatings (iMatrix-511, h-ESC Matrigel, or Laminin). The arrays were serially scanned starting 4 hours post-cell seeding and every 24 hours after to monitor clone formation.
Lessons Learned
Ultimately, the ability to screen tens of thousands of iPSCs on a single consumable using a fully integrated software and instrument platform to identify and isolate iPSC clones of interest has the potential to greatly increase the utility of iPSCs, improve iPSC characterization, and decrease the time to cell generation and therapeutic discovery.
For more on iPSC lines, view Dr. Hartman’s RaftNote: Taking the fear out of iPS cell line development.
Jessica Hartman, Ph.D.
Sr. Director, Applications at Cell Microsystems
Dr. Jessica Hartman has a B.S in Biology from the University of Virginia, a Ph.D. from Duke University in Molecular Cancer Biology and postdoctoral training in Biochemistry and Cancer Biology at Baylor College of Medicine and Duke University, respectively. She has previously served in Director-level roles, managing bioscience research and development for biotechnology companies At Cell Microsystems, Dr. Hartman’s role is to lead the development of new and streamlined workflows using the CellRaft Technology and its associated products.
