Recent scientific advances in stem cell research have provided enormous potential for the replacement or repair of defective tissues affected by trauma or diseases. Pluripotent Stem Cells (PSCs) based technologies could offer the potential for regeneration of patient/disease specific tissues in vitro, to enable improved understanding and treatment of inherited and chronic diseases and could even provide alternatives tissue sources for patients who would otherwise have to wait for tissues or organs to become available through traditional transplantation procedures. PSCs are of paramount importance to understand the onset and progression of human diseases, the ability to cure diseases thus optimising the outcomes of our health. PSCs are fundamental in toxicology studies and product safety assessments, drug testing and development of novel therapeutic strategies. Particular interest is being placed on iPS (induced pluripotent stem cells) by virtue of their enormous potential to model a variety of human cell types and eventually in modelling human conditions in cell cultures, in line with the recent advances in personalized medicine and pharmacogenomics.
ISENET has developed standard operating procedures (SOPs) to expand and characterize human, murine and ovine PSCs, by participating in a number of European financed projects. This includes performance of in vitro and in vivo differentiation, quality control testing such as viability, mycoplasma screening, microsatellite-based identity matching to the parent cell line, and karyotype analysis.
ISENET offers a number of services, performed under stringent quality control procedures, and each characterized cell line is accompanied with a Certificate of Analysis upon completion.
- Cell Culture: Culturing condition optimization: in vitro culture in adherence, onto extracellular matrixes coated vessels and in suspension (spheres)
- Cryopreservation and cell viability after cryopreservation: Storage: 24/7 monitored liquid nitrogen (-196°C) and nitrogen vapour phase (-150°). GMP-compatible protein-free freezing medium DMSO-based (Cryostor®; BioLife Solution). Trehalose-based (Patent pending) no-toxic cryopreservation medium (Treahalose: ISENET solution).
- Cell Authentication/Identification: Microsatellite analysis (STR and HLA)
- Contamination Testing: mycoplasma, bacterial and viral contaminant detection
- Cytogenetic Services: Karyotype assessment and cytogenetic diagnostic methods (SNPs) and high resolution analysis (aCGH). Extensive in vitro culturing over longer periods can lead to genetic and epigenetic changes that can alter cellular growth properties and cell lineages.
- Telomere Analysis: Telomere length and telomerase activity as indicators of pluripotency, self-renewal and proliferation. Telomere length by fluorescent in situ hybridization (FISH) Telomerase reverse transcriptase (hTERT),telomerase activity (TRAP assays), telomere restriction fragment (TRF) length analysis to evaluate the length of telomere and H2ax focus formation as a sign of DNA damage response (DDR).
- Cell Reprogramming:
- Cell Characterization: Assessment of pluripotency through marker expression via: real time PCR, FACS and immunofluorescence microscope.
- Cell Differentiation: Assessment of differentiation through marker expression via: real time PCR, FACS and immunofluorescence microscope.
- Neural Differentiation
- Pancreatic Progenitor Generation: In the developing pancreas, emergence of endocrine and exocrine progenitor cells from the ductal epithelium is a multi-step process that involves cell growth, migration into the surrounding mesenchyme and differentiation. Understanding the molecular mechanisms regulating islet cell growth and differentiation is of critical importance, and may ultimately result in the establishment of new technologies for the generation of a suitable insulin-secreting cell mass for transplantation in diabetes.
- Mesenchymal Stem Cell. Mesenchymal Stem cells (MSC) have an extensive replication capacity and the potential to differentiate into skeletal cell lineages as bone, cartilage and adipose cells. These characteristics along with their easy accessibility make them one of the best candidates for clinical applications and tissue replacement surgeries to regenerate bone and articular cartilage or cardiac muscle.
- Custom Tissue and Cell Microarrays:
- Nucleic acid Extraction: quality controlled DNA, RNA and miRNA suitable for Genome-wide analysis, Complete exome sequencing, RNA sequencing, Whole genome methylomes, Whole genome miRNA”omas”.
- Animal Science: embryology and organ dissection
- Organotipic Cell line derivation: fibroblast, myoblast, splenocytes, lung cells, pancreatic islets, neuronal cultures, neural stem cell, embryonic stem cells and induced pluripotent stem cells (iPS).
- Lymphoblastoid cell line derivation
- Genetic Analysis: DHPLC high throughtput mutation screening