Our Innovative Technology

The Process

1. Enrollment

• Membership registration and payment are conducted online or at the company office. CRYOkits are delivered to the customers/dental clinics for pre-assessment (4 deciduous incisors / 2 wisdom teeth). Afterwards, quantitative and qualitative reports on DPSCs & SHED analysis will be returned to customers. More teeth can be banked once passed the pre-assessment (maximum availability: 8 incisors / 4 wisdom teeth).

2. Tooth extraction and collection (E&C)

• Two streams of tooth extraction"

(A): Extraction at dental clinics,

(B) Home-extraction.

• Children aged 5-14: Deciduous incisors are extracted for SHED; Both (A) & (B) are applicable.

• Young adults aged 14-25: Wisdom teeth are extracted for DPSCs; Only (A) is applicable.

3.1 Extraction at dental clinics

• Professional dentists in cooperation can aid teeth extraction (both deciduous incisors & wisdom teeth) following strictly to the E&C protocol. CRYOkits are pre-deposited in dental clinics. This 8 extraction method is preferred with better sanitization and extraction skills for DSC integrity.

3.2 Home-extraction

• Customers can extract the wiggling deciduous incisors for their children at home conveniently using the CRYOkit. Extraction of wisdom teeth is unlikely due to the requirement of anesthesia. User guidelines and video demonstrations are available to assist the users in home extraction.

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4. Tooth packaging & shipment

• Extracted teeth (maximum of 2) are stored in one insulated jar at 4 °C and preserved in the tooth preservation cocktail & HBSS solution to prevent deterioration. If the customers do not have the CRYOkit on hand, storage of teeth in bovine milk at 4°C is also acceptable. Customers are required to deliver the packed CRYOkits to our laboratory via SF Express within 72 hours.

5. Isolation, digestion and cultivation of DSCs

• Isolation of DSCs for preservation by CRYOTeeth’s laboratories is possible up to 5 days after tooth extraction. Unprocessed teeth are preserved in HBSS solution and stored at 4°C.

• For digestion, teeth carrying the pulp-derived DPSCs and SHED are processed via mechanical and enzymatic (collagenase type I & dispose) digestion on the outermost enamel and adjacent dentin to expose the dental pulp and free the DSCs from the extracellular matrix.

• Once single-cell suspensions are obtained, they will be subjected to a 14-day incubation in MSC media. Multiple research suggests cell colonies were present and displayed viable cells after 14 days of isolation. For each tooth processed, 3⁄4 of the day-14 cultures are reserved for cryopreservation while the remaining 1⁄4 are subjected to cell analysis.

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6. Cell analysis

• Viability and cell count and DPSC and SHED Identification will be carried out.

Note: the samples have to pass BOTH the viability & cell count test and identification for pre-assessment.

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7. Storage

• We will employ cryopreservation below -195.8 °C on cryovials by liquid nitrogen vapor. Cell suspension will be placed in a preservation medium containing growth factors and a cryoprotective agent (CPA), dimethyl sulfoxide (DMSO) (Me2SO). DMSO inhibits the formation of ice crystals which may disrupt the cell membrane of DSCs.

• Controlled-rate freezing is applied until it reaches -90°C before it is transferred to liquid nitrogen vapor tanks. Significantly, studies have shown that post-cryopreserved DSCs remain competent 15 in MSC-hallmark trilineage differentiation, and a recovery rate of >85% was achieved.

• Individual reports are returned to each customer.