Collection, cryopreservation, and characterization of human dental pulp-derived mesenchymal stem cells for banking and clinical use

Abstract

Recent studies have shown that mesenchymal stem cells (MSC) with the potential for cell-mediated therapies and tissue engineering applications can be isolated from extracted dental tissues. Here, we investigated the collection, processing, and cryobiological characteristics of MSC from human teeth processed under current good tissue practices (cGTP). Viable dental pulp-derived MSC (DPSC) cultures were isolated from 31 of 40 teeth examined. Of eight DPSC cultures examined more thoroughly, all expressed appropriate cell surface markers and underwent osteogenic, adipogenic, and chondrogenic differentiation in appropriate differentiation medium, thus meeting criteria to be called MSC. Viable DPSC were obtained up to 120 h postextraction. Efficient recovery of DPSC from cryopreserved intact teeth and second-passage DPSC cultures was achieved. These studies indicate that DPSC isolation is feasible for at least 5 days after tooth extraction, and imply that processing immediately after extraction may not be required for successful banking of DPSC. Further, the recovery of viable DPSC after cryopreservation of intact teeth suggests that minimal processing may be needed for the banking of samples with no immediate plans for expansion and use. These initial studies will facilitate the development of future cGTP protocols for the clinical banking of MSC.

FIG. 1.

Schema for initial tooth processing. Forty human molars were placed in PBS and processed immediately after extraction to determine the efficiency of establishing DPSC cultures. All 31 cultures established were expanded and frozen at passages 2–3 for later cryopreservation/thawing studies. Eight cultures were randomly chosen for further characterization.

FIG. 2.

Schema of time-course experiments. Fifty-four teeth (different from those in Fig. 1) were placed into one of three collection/storage media and stored at 4^°C for 0–120 h before processing. DPSC from each culture were counted after 14 days.

FIG. 3.

Appearance of early DPSC cultures. Appearance of one representative DPSC culture 1 day (upper left panel, 40× magnification), 4 days (upper right panel, 20×), 6 days (lower left panel, 10×), and 10 days (lower right panel, 10×) after plating.

FIG. 4.

DPSC cultures express appropriate surface markers. Passage 3–4 DPSC from eight independent cultures were stained with antibodies helpful in defining MSC²¹ and analyzed by flow cytometry. Results from one representative DPSC culture are shown. The open histograms signify staining with isotype controls, and the filled histograms represent staining with the specified surface marker antibody.

FIG. 5.

DPSC have the potential to undergo osteogenic, chondrogenic, and adipogenic differentiation. Passage 3–4 DPSC were cultured in osteogenic (A,20× magnification), chondrogenic (B, 10 × magnification), or adipogenic (C,40× magnification) differentiation medium for 3 weeks before appropriate staining as described in the “Materials and Methods” section. Eight DPSC lines were tested, and all were capable of undergoing trilineage differentiation. Shown are photographs of one representative DPSC line. Color images available online at www.liebertpub.com/ten.

FIG. 6.

DPSC can be obtained from teeth transported in HTS, medium, or saline up to 120 h postextraction. Extracted teeth were placed in either PBS, HTS, or MesenCult basal medium and stored at 4^°C for various periods until processing. DPSC lines were established from teeth stored in all three collection/transport media after as long as 120 h postextraction. Shown here are DPSC cell counts 14 days after the cultures were established. n = 18 teeth for each collection/transport solution, 3 teeth per each time point. *p < 0.039; ^†p = 0.0008.