Human Induced Pluripotent Stem Cells (HiPSC):
Frequently Asked Questions
Is HiPSC culture the same as regular cell culture?
While the two process share commonalities, HiPSC culture differs from regular cell culture in several aspects. For instance, HiPSC grow poorly on bare plastics, so tissue culture vessels need to be pre-coated with extracellular matrices before HiPSC are plated. We provide HiPSC Coating Solution, a pre-diluted, ready-for-use preparation of growth factor-reduced basement membrane components, extracted from the mouse Englebreth Holm Swarm (EHS) tumor (analogous to Matrigel®). Also, HiPSC media should be kept at ambient (room) temperature of approximately 25oC rather than 37oC in a water bath, as it contains cytokines that are sensitive to warm temperatures.
From which tissue are your HiPSC isolated?
Our HiPSC are generated by genetic reprogramming of human dermal fibroblasts (HDF), located at the basal layers of the epidermis (skin). For custom HiPSC generation of unique donor profiles, genetic mutations or diseases, our scientists can isolate HDF directly from specific tissue (sourced by us or supplied by you). Alternatively, you can specify or send us your pre-isolated cells from which we make the HiPSC.
What are integration-free HiPSC?
Integration-free HiPSC are obtained through technologies that don’t involve the integration of vector and reprogramming transgenes into the genome of the target cell. Specifically, the Sendai virus is an RNA virus that never goes through a DNA phase, thus there is no risk of random integration of viral genetic material in the host genome. Thus, our integration-free HiPSC ameliorate the need to deeply scrutinize phenotypes observed as potential artifacts of random integration.
Is the Sendai virus still present in HiPSC?
The Sendai virus used to generate HiPSC is replication-deficient. Therefore, in spite of being very efficient at infecting host cells, reprogramming them towards HiPSC, its propagation abilities are limited, and viral load decreases significantly as HiPSC divide and are propagated. Nonetheless, residual virus presence may be detectable in lower passage HiPSC. All personnel working with HiPSC must be fully trained in the use of appropriate tissue culture techniques, always assuming that pathogens may be present in the cultures.
What is the importance of Karyotyping HiPSC?
Karyotype abnormalities such as chromosome gain, loss or translocation of a part of a chromosome are a common occurrence in cell culture. Ensuring that HiPSC have a normal karyotype is fundamental to warrant that cell behaviors, observed phenotypes or test drug effect is genuine and not an artifact of abnormal karyotype. Given the rapid growth kinetics of HiPSC, they are susceptible to those abnormalities. In order to minimize the chances of karyotype abnormalities experiments using HiPSC should be performed through a maximum of 40 passages, and it’s recommended that karyotype analysis is conducted at every 20 passages.
What is the doubling time of HiPSC?
The growth behavior of earlier passage HiPSC is dynamic, which is an intrinsic characteristic of HiPSC. As a consequence, the doubling time of HiPSC lines is not very informative, as it changes over the time that the line is sub-cultured. It is not uncommon that lower passage HiPSC need to be propagated with lower split ratios, while HiPSC cultured for a larger number of passages may present a faster growth. It is also important to note that the format of HiPSC culture (colonies vs. monolayer), the HiPSC media as well as the extracellular matrix used can all impact HiPSC growth kinetics.
What kind of medium should I use for these HiPSC?
Our goal is to provide the very best media, facilitate technical support, and reduce variables when troubleshooting -- topics pertinent to HiPSC. As such, Cell Applications, Inc. guarantees cell performance and provides warranty replacement when cells are cultured with CAI media & reagents. Our HiPSC are expanded in Human Induced Pluripotent Stem Cell (HiPSC) Growth Medium, available from Cell Applications, Inc., [Certain similar formations go by the name "Essential 8TM (E8)" medium]. Likewise, when thawing and starting a culture of HiPSC to perform initial propagation, our instructions call for the use of CAI's HiPSC Growth Medium, as well as HiPSC Coating Solution for culture ware (a preparation analogous to Matrigel®). If protocols or situations deman, customers may utilize media from other manufacturers, as long as several passages are included for the cells to adjust to the new medium.
What is ROCK Inhibitor, and why should it be used with HiPSC?
ROCK is an abbreviation for Rho-Associated Protein Kinase. Y-27632 is a small molecule inhibitor selective for ROCK1. Targeting ROCK1 is known to increase stem cell survival & cloning efficiency, without affecting pluripotency.
What do you mean by “1 Billion – 1 Lot – 1 Week?”
With unprecedented scale, large quantities of HiPSC can be generated through our on-demand HiPSC production and expansion services. For example, over one billion HiPSC from one lot can be expanded in a week. Depending on the project scope & cell type, a little extra time for QC may be needed.
Compared to your method, how long would it take my own lab to make a billion cells?
An estimated 40 human hours (spread across multiple weeks) would be needed to make a billion cells the traditional way. The work would require at least 3 highly trained people, and regular personnel turnover means even more time is wasted in training. Another bigger issue is consistency across the batch. Traditional methods equate to thousands of manual maneuvers and have many opportunities for mistakes and contamination. We provide the cells from a single lot of material, with many fewer steps and much less hands-on time. Bottom line, laboratory staff report spending 30 to 80% of their time in manual labor generating and tending to HiPSC the "usual ways". Wouldn't you rather have them running meaningful, value-added experiments?
You've compared HiPSC technology with the computer industry. How so?
Robert Petcavich, Co-founder at our partner StemoniX says, "we view stem cells as chemical microprocessors. Instead of processing electrons like computer chips, cells compute chemical information based in the DNA. So we have borrowed micro manufacturing techniques and processes from the semiconductor industry to scale cell production. We use highly automated manufacturing equipment in a very clean and sterile environment, which has been modified for our protocols to produce billions of cells per week. Because we produce the cells all at the same time the cell-to-cell quality is identical which is important when screening for drug compounds to maintain data fidelity. Our patent-pending manufacturing details are proprietary and allow us to be a low cost producer."
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