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Stem Cells Safety

Stem Cell Safety

QC Activities in the Biosafety Laboratory of EmProCell

The Biosafety Laboratory of EmProCell is uniquely designed to facilitate screening and detection of isolated & purified stem cells for various bacterial & viral pathogens as per the Guidelines for Stem Cell Research and Therapy jointly written by Dept. of Biotechnology, Govt. of India and ICMR. The laboratory is outfitted with cell culture and molecular biology equipment and further includes ELISA Lab, cell culture lab, pathogen detection lab and karyotyping lab. The QC activities performed by the Biosafety Laboratory are as enlisted below,

Bacterial Endotoxin Testing

Endotoxins are potentially toxic natural compounds present in the cell wall of gram negative bacteria. This material is pryrogenic meaning that it can cause high fevers in humans. It can also induce acute febrile shock when present in miniscule amounts.

The bacterial endotoxin is detected or quantified using Limulus Amoebocyte Lysate (LAL) which is an extract of blood cells from the horseshoe crab (Limulus polyphemus). 

The sample (abortive material) received from hospitals is washed with the washing solution to remove blood impurities and tissue debris. The washed fluid is received from the manufacturing laboratory for endotoxin detection. The presence of endotoxin is detected by LAL test using Chromogenic Endpoint Assay. Samples containing endotoxin limits above 0.125 EU/ml are considered as positive samples and are discarded immediately. Endotoxin free samples are further processed by the Manufacturing Department for separation of the fetal organs & tissue extracts and isolation of stem cells from them.

Microbial Sterility Testing

The collection, transport and processing of fetal material or tissues always carry a significant risk of microbiological contamination.  The Food and Drug Administration requires that all parenteral biological products undergo sterility testing using the compendial sterility method to ensure that products such as vaccines, tissue extracts and cells are safe when they reach the recipient. This microbial sterility testing method is based on the observation of turbidity in liquid culture media due to the growth of bacteria (aerobic & anaerobic), yeast and fungi At EmProCell, aliquot of isolated stem cell preparation is used for microbial sterility testing using BD Bactec instrument. The samples are incubated in culture bottles for 7 and 14 days to check for bacteria and fungi contamination respectively. If the sample fails the microbial sterility testing, than all the other cryopreserved aliquots of the sample are discarded. Only aliquots of samples that pass the microbial sterility testing are further processed for detection of various infectious pathogens. 

Pathogen Detection using Real Time PCR

Documentation of complete history of the cells and their characterization, for use in therapy is essential to safeguard against potential risks of biological therapy. This is particularly important when allogenic cells are used for this purpose. At EmProCell, all the stem cell preparations and the tissue extracts are tested for panel of viral and bacterial pathogen in order to avoid unexpected transmission to the recipient.

The samples which clear the microbial sterility test are further used detection of pathogen using most sensitive techniques like PCR and RT-PCR techniques. The pathogens detected includes, HBV, HCV, CMV, EBV, HSV1 &2, HIV 1 & 2, Treponema pallidum Mycoplasma hominis, Ureaplasma sp., Chlamydia and Toxoplasma gondii. For this, nucleic acids (DNA & RNA) are isolated from the cell samples, quantified and then used for pathogen detection using Nucleic Acid Amplification Assays. Only aliquots of samples that pass the pathogen detection test will be further processed for karyotyping. The isolated cells are also tested for X and Y chromosome using Real Time PCR.

Karyotyping

The samples are further karyotyped for detection and identification of chromosomal abnormalities in the stem cells. 

The cells that qualify all the above criteria will only be used for the future therapeutics, rest of the cells are discarded.