Are you confident that your cell lines and biological models are really what you think they are? Do you use authenticated cell lines in your experiments? Do you check cell lines regularly? If not, then you should! Cell culture development has significantly changed the area of life sciences and contributed to great advancements in medicine. Research using cell lines is essential for modeling diseases, stem cells, cancer investigation, and establishing therapies. However, did you know that an estimated 18-36% of cell lines are misidentified, cross-contaminated, or genetically drifted with other cell lines? In the past, karyotyping & isoenzyme analysis were used for cell line authentication. However, these techniques needed to be more reliable to distinguish between cell lines. In contrast, STR profiling was reliable, low-cost, and easy authentication method for cell lines. If applied routinely in cell culture management can significantly improve the cell line defects resulting in more accurate assays.
Table of Contents
What is the Purpose of Authenticating a Cell Line?
Scientists are not easily fooled, but they can unwittingly fool themselves. For example, the identity of a cell line might not be what they assume it is. The following are some possibilities that occur due to a lack of accurate cell line authentication tests:
- Often, a cell line of interest is misidentified at the source. As a result, it could have led the researcher to use the same (misidentified) cell line to conduct the research, ultimately not getting the desired result.
- A second issue with cell lines is the possibility of cross-contamination, where the contaminant can be a different cell from another source/sample, environmental pollutants, etc.
- The third and most vital problem with cell line authentication is that they often become genetically drifted with time. A cell may lose its originality and become a different cell during genetic alteration. Researchers and experts generally acknowledge that the latter is more harmful than the first two types of errors, as one may need to realize that the cells are gradually changing and may continue to get false results.
Therein, Short Tandem Repeat (STR) DNA profiling is one solution that has been proposed for the detection of misidentification and other types of errors in human cell lines for accurate results.
When to Authenticate Your Cell Line?
When a lab receives a cell line, it should obtain all the relevant information about its origin, growth characteristics, and culture conditions. Recommended intervals for STR profiling for cell line authentication are:
- When a new cell line is derived
- When a cell line is acquired from a non-repository source
- Before initiating new experiments
- Before submission of research work for publications
- When a change in morphology and characteristic behavior of the cell is observed
Method of a Cell Line Authentication DNA Test
The STR profiling method is used for a cell line authentication test in India. ‘STR’ stands for ‘short tandem repeats. These are a few base-pair long repeating sequences on the DNA molecule and are used for simplicity and ease in the authentication process.
You can give the sample either in the form of a cell pellet or extracted DNA.
Extracted DNA-
- Samples should be collected in 1.5-2.0 ml microcentrifuge tubes with proper labeling.
- Extract and purify the DNA by one’s convenient method, finally eluting it in Low TE or Nucleolus Free Water. Concentration should be 10-100ng*/µl. DNA Quantification should be done with a qubit fluorometer, not a Nanodrop spectrophotometer, and the volume of each tube should be 10-20 µl.
- Seal the tubes properly with paraffin.
- Freeze the tubes at -20°C
- Put the tubes into a zip bag or box. Ship the sample with ice packs.
Cell pellet-
- The sample should be collected in 1.5-2.0 ml microcentrifuge tubes with proper labeling.
- Protocol for attached cells – Trypsinize the cell culture and then centrifuge it with 125 x g. Discard the supernatant and resuspend the pellet into PBS** to make the concentration 1.6 x 106 cells/ml. Confirm the final concentration by cell counting. If it’s more diluted, repeat the process with a low volume of PBS.
- Protocol for suspension cells – Collect and count the cells for the optimum concentration of 1.0 x 106 cells/ml. If it’s highly concentrated, then dilute with PBS. If cells are already more diluted, then centrifuge and resuspend into PBS to make concentration optimum.
*ng – nanogram
**PBS (Phosphate Buffered Saline) – It is a non-toxic solution. Unlike water, PBS prevents cells from rupturing or shriveling due to osmosis.
Detection Method
In the PCR reaction, fluorescent primers recognize different loci. When the PCR is complete, the internal standards for size can be added to the mixture, and the DNA can be separated by size using capillary gel electrophoresis.
Size analysis is then completed using the GeneMapper ID-X (GMIDX) software, which compares the size of different DNA fragments with internal standards. Then, the short tandem repeat (STR) genotyping is done by converting the amplicon sizes into alleles using the allelic ladder.
Characteristics of Authenticating a Human Cell Line
- Modified genetic makeup compared to the donor
- Easily reproducible models
- Provides consistent results
- Immortalized (unlimited life span)
- Not usable as an in-vivo model
- Lower physiological relevance
- Possibility of sub-culturing
DNA Forensics Laboratory – For Best Cell Line Authentication Services in India
You can outsource this tedious work of cell line authentication tests in India to DNA Forensics Laboratory Pvt. Ltd. We offer complete cell line authentication test services using STRs profiling. This enhances researchers’ abilities to detect misidentifications, genetic drift, and cross-contamination of cell lines.
Research Institutes, Pharmaceutical Companies, and Biotechnology Companies are our major clients. Moreover, we are the only company that offers legal DNA tests in India. With 400+ collection centers across India, you can visit your nearest collection center to give your DNA sample.
Other key factors that make us desirable are:
- Latest and advanced methodologies for the test
- Highly qualified and experienced technical staff
- Providing NABL-DNA testing facility
- Fastest turnaround time of 10-15 business days
For cell line authentication, the client has to send samples directly to our testing facility in New Delhi. For further queries about Cell Line Authentication Tests Services in India, get in touch with us at +91 8010177771 or WhatsApp at +91 9213177771 to schedule an appointment.