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From the Umbilical Cord to the Cryo-preserver – Part 3
In this multi-part series, part 2 focused on the processing of cord blood samples in the LifeCell lab, Chennai. In part 3, we will look at the various stages involved in the processing of cord tissue sample under sterile condition.
Cord Tissue Sample
The cord tissue refers to the long cord that connects the mother and the baby and contains mesenchymal stem cells. The technique involved in cord tissue processing is extensive and different from that of cord blood processing. During collection, the cord tissue is cut into 2 long pieces, each measures about 10-12 cms long and placed in 2 different tubes.
While talking to Mr. Ramesh, Product Manager – Cord Tissue at LifeCell, he said “… cord tissue is sticky in nature, so it is prone to contamination. We maintain aseptic sterile environment to process the samples and follow strict protocols to keep them contamination free”. An entire team of experienced technicians handle the cord tissue processing efficiently on time. “Every day, we have 3 shifts with 12-15 members in each shift to process the samples on time”, he added.
The cord tissue processing involves a long set of procedures to isolate the stem cells, which are as follows:
Preparing Cord Tissue Samples for Processing
Before processing, the samples undergo the following steps:
In the sample receipt area, the details present in the purple card inside the collection kit box will be cross verified; the CRM number and kit box number will be checked with the database.
The samples including cord blood, cord tissue and maternal blood are provided with a bar-coded lab ID for easy access and processing, which will also avoid the mixing up of samples.
Cleaning of the Sample
The cord tissue sample is collected in 2 tubes with 10-12 cm of cord tissue in each. The outer surface of the tubes is cleaned with isopropyl alcohol to avoid any contamination before removing the parafilm seal.
Processing of Cord Tissue Sample
After cleaning, the samples are moved to the cold room which has a temperature of 2-8 degree Celsius. The samples are then washed with antibiotic solution to prevent contamination; as the solution has validated concentration of antibiotics, it does not harm the mesenchymal stem cells present in the cord tissue.
The samples are moved to the processing room and again washed with antibiotic solution for the second time to ensure maximum protection against contamination. The processing room maintains a strict aseptic environment based on the ISO quality standards with UV sterile laminar air flow (processing workstation), sterilized equipments etc.
Here, the samples are divided into 3 parts: one part is preserved in the form of tissue for future use ; the second part is used for the proliferation of mesenchymal stem cells through explant tissue culture technique; the third part is preserved as a back-up, which will be used if need arises.
Cord Tissue Preservation
One part of the sample is kept in a petri-plate and cut into small pieces that measures about 2-4 mm3 in size. Autologous plasma derived from the same umbilical cord blood is added to the small pieces of cord tissue. A cryoprotectant solution is added to the mixture to maintain the viability of cells during cryopreservation.
Culture of Cord Tissue Stem Cells
The stem cells of cord tissue are cultured using explants culture technique to increase the number of cells. The second part of the cord tissue is cut into pieces and added to 5ml of culture medium. This culture is kept in an incubator at 37 degree Celsius for 25-30 days which allows the cells to proliferate.
The culture media is frequently changed under sterile conditions to provide fresh nutrients for the cells and to remove debris. The growth is frequently monitored under microscope to detect the growth nature of stem cells and also to check for microbial presence. During cell harvest, the mesenchymal stem cells are isolated from the cell culture plate and counted using automated cell counter. The medium will be tested for contamination and cell growth. If the stem cells are free of contamination and shows desired amount of cell growth, they are added to cryovials and moved to the cryo-preservation area.
At LifeCell, the technicians make sure that the samples are 100% pure and yield maximum proliferation of viable stem cells before cryopreservation.
If the culture shows contamination, the third part of the cord tissue will be tested, treated and used for a new culture batch. In the next part, let us check out the procedure involved in testing the mesenchymal stem cell culture.