Laboratory FAQ

The dental pulp is processed in Class 100 clean room in the Lifecell facility at Chennai. Class 100 clean room means that there are less than 100 particles with diameter of more than 0.5 microns. Normal atmospheric air has more than 35,000,000 particles of more than 0.5 microns

The quality of the stem cells is greatly dependent on the sterility of the dental pulp and hence due care is taken that the extracted dental pulp does not get disinfected during processing.

After the tooth reaches the laboratory, it is taken to the clean room for processing. There are 4 steps involved in the processing of the dental pulp:

• Disinfection of the collected tooth: The collected tooth is disinfected by flooding it with an anti-infective – Chlorhexidine and then submerging it in chlorhexidine for a couple of minutes.
• Breaking open the collected tooth: The disinfected tooth is opened using a piezoelectric machine
• Extraction of the dental pulp from the tooth chamber: The dental pulp is extracted from the opened tooth using the excavator
• Dissociation of the dental pulp: The extracted dental pulp is then dissociated in DPBS (Dulbecco’s Phosphate Buffered Saline)

To maintain the viability of the dental pulp the tooth is transported at temperatures between 2 – 8 deg. It is important that the temperature of the preservative medium (penicillin & streptomycin) should not go below 0 deg as the water within the preservative medium freezes and damages the dental pulp

The kit box contains a thermobutton which records the temperature every half an hour. On reaching the laboratory the readings from the thermobutton are downloaded to check the temperatures throughout the transfer of the tooth from the dentists clinic to the laboratory. A thermobutton can record 2000 readings at any given period of time. It can be reprogrammed and hence can be recycled.

The preservative medium of penicillin and streptomycin is used during transportation to prevent infection and drying. The preservative medium is of a special quality which can withstand stability at lower temperatures. The medium is imported from France.

The presence of mesenchymal stem cells is confirmed using an analytical equipment called RT PCR. This is one of the quality control tests performed on the dental pulp. The presence of the stem cells is verified using biomarkers and the readings are interpreted in terms of Ct values i.e. number of cycles to arrive at the signal detected by the machine.

We reject the sample under two conditions:

 

• When there is no signal detected: When there is no signal detected by the machine, it is inferred that there are no stem cells in the sample and hence the
• Signal is detected but the Ct value is more than 33: When the signal is detected, it indicates the presence of the mesenchymal stem cells but since the Ct value is more than 33, we reject the sample as we infer that the stem cell number is lower than the stipulated

 

In both these situations, the test is repeated 3 times to rule out any error in measurement and only then the sample is rejected and conveyed to the client.

In very rare situations, if we find that the dental pulp is infected during the sterility tests then the sample is cryopreserved but quarantined in paper bags. The infected organism is also detected and records maintained in such a situation.

We use agar plates for sterility testing and tests are done for both aerobic as well as anaerobic organisms

We do serology tests on the collected blood sample to check out whether the client is suffering from any of the following infections:

 

• HIV(Human Immuno Defeciency Virus),

 

• HBV(Hepatitis B Virus),

 

• HCV(Hepatitis C Virus),

 

• CMV(Cytomegalo virus),

 

• Syphilis and

 

• Malaria.

If the blood sample tests positive for HIV, the tests is reconfirmed from an external agency and if is found to be positive we do not store the sample and the client is communicated

If the CMV test is positive there is no need to panic as it is very common to have one. One pager explanation is given along with the certificate to sort out all the client doubts

One school of thought says that it is better to preserve the dental pulp as the therapy might demand different growth factors to be used during expansion and differentiation. Hence we maintain the dental pulp now. However requisite validation runs have been done in India to verify the expansion of the stem cells

We store the dental pulp submerged in a cryoprotectant DMSO distributed across 5 cryovials within the cryotank. The cryovials are made up of polypropylene and can withstand extreme conditions. A cryoprotectant is a substance that is used to protect the dental pulp from freezing damage.

Theoretically we can store the dental pulp at low temperatures (less than -150 deg) over 40 years.

Liquid nitrogen is used to maintain the low temperatures in the cryotanks. The cryovials are stored in the vapour phase. This vapour is generated from the liquid nitrogen stored at the bottom of the tank. When the liquid nitrogen goes below the stipulated levels, the temperature starts rising which is monitored 24/7 and replenished regularly

The cryovials never come in contact with the cryotanks and hence no contamination. Further on no organisms can remain alive at such low temperatures to cause any contamination

Tests have been done to check out the presence of stem cells on retrieval. Dental pulp stem cells have one of the best retrieval rates. i.e 93% – 93% of the cells were alive even after cryopreservation

For retrieval, the client has to write to Stemade along with the details of the doctor who is going to use the cells and prescription from the doctor. Further on details for the purpose of retrieval and all regulatory policies need to be complied with

Typically;

 

One dental pulp           = 1.2 ml of digested dental pulp

 

= 1 million cells live cells

 

= >100,000 mesenchymal (10-20% of total cells)

 

First of all, most of the clinical application using stem cell are starting from about 1 or 2 millions cells after expansion. It as been demonstrated that the conservation of expanded dental stem cells could be done with a concentration per vial of 0.5 to 2 millions cells/vial (Woods, 2009). AT this concentration, the average post-thaw viability is > 93%. Suchanek, have cultivated undifferentiated DPSCs for long time, over 60 population doublings in cultivation media designed for bone marrow MPCs. Initial doubling time of our cultures was from 12 to 50 hours for first 40 population doublings, after reaching 50 population doublings, doubling time had increased to 60-90 hours.

Stemade has the capacity to expand the stem cells but this will invite a separate costs which needs to be discussed at the time of retrieval. The cost will depend on the number of stem cells required as the number differs with the therapy / ailment.

This cannot be disclosed as it is patented. Typically contains alpha MEM (Minimum essential medium), ascorbic acid, glutamine, penicillin, streptomycin

The preservation certificate gives 4 key details:

• Size of the dental pulp : the size is good indicator of the number of live cells. Big, medium and small is relative to the average size of the dental pulp in India which has been derived from the validation samples
• Presence of CD plus cells : Confirms the presence of mesenchymal stems cells in optimal quantities. The count is not specified as it has no relevance in dental stem cell banking as the cells have to be expanded for therapy. In umbilical cord it is important to mention the count as they cannot be expanded.
• Microbiological sterility tests: confirms the absence of infection. If any infection is detected the bacterial id is given
• Serology testing results: confirms absence of infectious diseases like HIV, etc.