Optimized cryopreservation method for human dental pulp-derived stem cells and their tissues of origin for banking and clinical use

Abstract

Dental pulp is a promising source of mesenchymal stem cells with the potential for cell-mediated therapies and tissue engineering applications. We recently reported that isolation of dental pulp-derived stem cells (DPSC) is feasible for at least 120 h after tooth extraction, and that cryopreservation of early passage cultured DPSC leads to high-efficiency recovery post-thaw. This study investigated additional processing and cryobiological characteristics of DPSC, ending with development of procedures for banking. First, we aimed to optimize cryopreservation of established DPSC cultures, with regards to optimizing the cryoprotective agent (CPA), the CPA concentration, the concentration of cells frozen, and storage temperatures. Secondly, we focused on determining cryopreservation characteristics of enzymatically digested tissue as a cell suspension. Lastly, we evaluated the growth, surface markers and differentiation properties of DPSC obtained from intact teeth and undigested, whole dental tissue frozen and thawed using the optimized procedures. In these experiments it was determined that Me₂SO at a concentration between 1 and 1.5 M was the ideal cryopreservative of the three studied. It was also determined that DPSC viability after cryopreservation is not limited by the concentration of cells frozen, at least up to 2 × 10⁶ cells/mL. It was further established that DPSC can be stored at −85 °C or −196 °C for at least six months without loss of functionality. The optimal results with the least manipulation were achieved by isolating and cryopreserving the tooth pulp tissues, with digestion and culture performed post-thaw. A recovery of cells from >85% of the tissues frozen was achieved and cells isolated post-thaw from tissue processed and frozen with a serum free, defined cryopreservation medium maintained morphological and developmental competence and demonstrated MSC-hallmark trilineage differentiation under the appropriate culture conditions.

Introduction

The term “stem cell” generally refers to a cell possessing the ability to self-replicate and give rise to daughter cells which undergo an irreversible, terminal differentiation process [2], [10]. Stem cells from post-natal human origin have been studied extensively from sources such as the epidermis, gastrointestinal epithelium, adipose tissue, umbilical cord blood and bone marrow. To date, the hematopoietic stem cells originating in the bone marrow have arguably been the most extensively studied [13]. Also first identified in the bone marrow is a population of multipotent mesenchymal stromal cells (“mesenchymal stem cells” or MSC) which contribute to the formation of multiple mesodermal tissue types, such as bone, muscle, cartilage, ligaments, tendons, and adipose tissue [4]. MSC or MSC-like cells have now been isolated from various tissues and are of interest to many researchers and clinicians due to their easy isolation, ability to greatly expand in culture and many potential uses in cell-mediated therapies and tissue engineering applications [8].

One source from which MSC have not been as thoroughly studied is dental tissue. Recently, several groups have initiated investigation of this potential source and have begun to examine the properties of stem cells isolated from dental pulp, periodontal ligament and periapical follicles of adult teeth, as well as from deciduous teeth [8], [9], [12], [16], [19]. As these cells are characterized and their importance realized, reproducible methods for harvest, banking and distribution become critical.

Results of recently published studies on cryopreservation of dental-tissue derived mesenchymal stem cells have been promising, but are preliminary at best. To date the most successful cryorecovery procedures have used previously established cell cultures [14], [21]. However, while these studies demonstrated good recovery post-thaw, the cells were derived from relatively low numbers of teeth, and this method requires extensive up-front processing. One study evaluated cryopreservation of intact periodontal ligaments with processing and culture establishment post-thaw; however this group reported a great reduction in colony development as compared to controls [19].

Our group recently reported the first study in which the processing of dental pulp-derived MSC (DPSC) from harvest to storage was considered using large numbers of teeth [18]. In that study we reported that isolation of DPSC is feasible for at least 120 h after tooth extraction, and that cryopreservation of established early passage cultured DPSC leads to high-efficiency recovery after thawing. Furthermore, we demonstrated recovery of viable DPSC after cryopreservation of intact teeth, suggesting that minimal processing may be needed for the banking of samples with no immediate plans for expansion and use.

To optimize methods for banking DPSC, the optimum cryopreservation process should be straight forward and effective when applied to the tissue as a whole, with the idea that stem cells would be extracted post-thaw. The rationale for this is to preserve clinical samples for subsequent stem cell recovery, since immediate cryopreservation of tissues will be more practical than direct primary isolation of stem cells, which requires additional equipment and personnel [7]. Indeed, isolating DPSC can be laborious, time-consuming and expensive, especially while employing current good tissue practice (cGTP) standards for clinical use of the cells; therefore, cryopreservation of whole teeth or isolated tooth tissues may be advantageous for the banking of specimens from which DPSC cultures are not immediately needed. If cells are immediately needed from tissues, cryopreservation procedures need to be as efficient as possible to maximize the utility of the material.

In the present study, we now report results of optimized methods for cryopreservation and processing of DPSC and their respective tissues of origin for banking and to allow further study of the potential therapeutic uses of these cells. To that end, we further investigated additional processing and cryobiological characteristics of DPSC. First, we aimed to optimize cryopreservation of established DPSC cultures, with regards to optimizing the cryoprotective agent (CPA), the CPA concentration, the concentration of cells frozen, and storage temperatures. Secondly, we focused on determining cryopreservation characteristics of enzymatically digested tissue as a cell suspension. Lastly, we evaluated the growth, surface markers and differentiation properties of DPSC obtained from intact teeth and undigested, whole dental tissue frozen and thawed using the optimized procedures.