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1.What format will the cells be delivered to clients: frozen vials or pre-plated cells?
2. How can I contact Abcam if I have a question?
3. Are ioSkeletal Myocytes fully differentiated?
4. Can you propagate ioSkeletal Myocytes once received?
5. What seeding density do you recommend for the ioSkeletal Myocytes?
6. How are cells cultivated?
7. How soon after delivery can ioSkeletal Myocytes be used for experiments?
8. Why is opti-ox better than other methods of cellular programming?
9. What were the cells of origin for ioSkeletal Myocytes?
10. Do you use viral vectors to manufacture ioSkeletal Myocytes?
11. What is the host and transgene used to generate cells?
12. What quality control is performed on ioSkeletal Myocytes?
13. How do you confirm your cell lines are free from contamination?
14. Can you please provide some references for the ioSkeletal Myocytes?
Cells are provided as frozen vials, in either small (>2.5x106 viable cells) or large (>5x106 viable cells) sizes and shipped on dry ice. They should be immediately stored upon arrival in liquid nitrogen or ultra-low temperature freezers (-150oC) until use.
If you have a question regarding our products or services, you can contact our customer service and scientific support team here.
ioSkeletal Myocytes are not fully differentiated when the end-user receives them. ioSkeletal Myocytes are shipped as ‘primed’ skeletal myocytes that have been generated from human pluripotent stem cells using patented opti-ox cellular reprogramming technology. Cells are delivered in a cryopreserved format are programmed to rapidly mature upon revival in the recommended medium.
The protocol for cell generation is a three-phase process:
Figure 1: Schematic representation of the three-phase protocol to produce and culture ioSkeletal Myocytes.
ioSkeletal Myocytes cannot be propagated or passaged further in culture because they have initiated reprogramming.
Skeletal myocyte cultures are obtained by plating ioSkeletal Myocytes at a minimum seeding density of 100,000 cells/cm2. This may require optimization depending on the experiment and plate format. We do not advise seeding below 100,000 cells/cm2.
Cells are cultivated in serum-free, chemically defined culture conditions, as a 2D monolayer on Geltrex coated TC dishes. They are cryopreserved in knockout serum replacement (CTS-grade) supplemented with 10% DMSO.
ioSkeletal Myocytes are delivered in a cryopreserved format and are programmed to rapidly mature upon revival in the recommended media. By day three post-revival, ioSkeletal Myocytes demonstrate classical myocyte morphology and express the myocyte genes DES, MYOG, and myosin heavy chain, as assessed by quantitative reverse transcription PCR (qRT-PCR). By day seven post-revival, skeletal myocytes demonstrate expression of GLUT4 in peri-nuclear regions and striations. Skeletal myocytes express the major proteins of myofilaments, including myosin heavy chain, desmin, dystrophin, and troponin. By day 10 post-revival, form-striated, multinucleated myocytes that contract in response to acetylcholine are present.
ioSkeletal Myocytes are derived from human-induced pluripotent stem cells (hiPSCs) using proprietary opti-ox technology (as described by Pawlowski et al in 2017) which relies on the precise genetic engineering of hiPSCs with the transcription factor(s) defining a specific cell identity. The opti-ox system enables unprecedented batch-to-batch reproducibility, homogeneity of differentiation, and scalability compared to classical approaches using non-targeted transgenesis (eg viral vectors). ioSkeletal Myocytes are easy to culture, and within days of revival convert into homogeneous and mature skeletal myocytes.
ioSkeletal Myocytes are generated from hiPSCs. The parental iPSC line has been derived from Caucasian white male dermal fibroblasts using the four retrovirally transduced Yamanka factors:
No, only recombinant DNA vectors are used to generate ioSkeletal Myocytes from the parental hiPSC line*.
*However, replication-deficient retroviral vectors (non-infectious) have been used for the reprogramming of the parental hiPSC line (characterized master line from dermal fibroblasts).
The host is human, and the transgene used to differentiate the hiPSCs towards ioSkeletal Myocytes is MYOD1. The cells also express additional transgenes that are an integral part of the opti-ox system:
ioSkeletal Myocytes production batches are tested for sterility, viability, and maturity acquisition over time by monitoring the expression of key genes by RT-qPCR. This includes monitoring for loss of pluripotency (OCT4 and NANOG), and acquisition of Myosin Heavy Chains (MYH2, MYH3, MYH8), Troponin (TNNT1), along with Desmin (DES), Dystrophin (DMD) and the skeletal myocyte transcription factor Myogenin (MYOG). In addition, production batches are tested by immunocytochemistry for the expression of Myogenin, Desmin, Dystrophin and Myosin Heavy Chain. The genetic integrity of the parental hiPSC clone used for ioSkeletal Myocytes manufacturing is tested by G-banding karyotyping and array genome hybridisation (AGH). Absence of common human pathogens (Hepatitis B, Hepatitis C, HIV1, HIV2, HTLV 1, HTLV 2), mycoplasma, bacterial and fungal growth is confirmed by validated means.
We follow strict aseptic bio-banking procedures. Each manufactured cell lot is tested for sterility (microbial and fungal) and absence of mycoplasma infection (pan species) by industry-standard, validated means, post-thawing.
Please refer to the publication describing the reprogramming of human iPSCs into skeletal myocytes by MYOD driven opti-ox cellular reprogramming: