Human platelet lysate permits scale-up of dental pulp stromal cells for clinical applications
Introduction
The transplantation of human dental pulp stem/stromal cells (DPSC) in human mandible bone (1) has created a significant impact in the field of regenerative medicine. For the first time, this has moved dental stem cell research from the bench to the bedside. Human DPSC originate from a neural-crest lineage and can be obtained non-invasively from teeth that are extracted for clinical reasons and usually discarded as biologic waste (2,3). Studies have shown that DPSC are capable of differentiating into fully functional neuronal cells (4), odontoblasts (5), endothelium (5), hepatocytes (6) and pancreatic cells (7), and have shown some remarkable outcomes in pre-clinical studies (8,9). Thus, it will not be surprising if DPSC emerge as a potent surrogate for traditionally used bone marrow (BM) mesenchymal stromal cells (MSC) in human clinical trials. This paradigm shift opens avenues into the treatment of debilitating diseases with a tailor-made choice of stem cells in anticipation of achieving maximum efficacy with regenerative medicine. However, just like any other MSC source, the use of DPSC in transplantation requires large-scale expansion of cells in order to cater for the need for clinical quantity.
In most clinical trials, including the first human DPSC transplantation, fetal bovine serum (FBS) has been used as the main nutritional supplement. However, the use of xenogeneic serum is complicated because of high lot-to-lot variability coupled with the risk of transmitting infectious agents and immunizing effects (10,11). Therefore there is always a need to search for alternative sources to replace FBS, but these substitutes must show competitive results. A chemically defined xenofree medium would be the preferred solution; however, such a formulation that allows for both isolation and expansion has not been achieved thus far (12). In addition, making a chemically defined media as good as FBS would perhaps be more expensive than FBS itself, which ultimately would hamper production at large scales.
Conversely, human blood products, such as autologous and allogeneic human serum, human plasma, cord blood serum and human platelet derivatives, including platelet lysate (PL) and platelet-released factors (13., 14., 15., 16.), have been introduced increasingly in stem cell therapy as a compelling substitute for FBS. The buffy coat-derived platelets are of particular interest because they do not compete with erythrocyte and plasma preparations for the limited available blood donations (17). This is because the platelets are separated from the white and red blood cell fractions and can be concentrated to at least 1 × 109 platelets/mL by centrifugation. The release of growth factors and mitogens stored in alpha granules of platelets may be induced by platelet activation by thrombin or cell fragmentation during repeated freeze–thaw cycles (18). Among the persuasive mediators released from platelets are adhesive proteins, coagulation factors, mitogens, protease inhibitors and proteoglycans (19). Hence, human platelet lysate (HPL) may replace FBS in many cell culture systems that have previously been thought to be solely dependent on the presence of FBS.
Although much of the past literature has focused on BM MSC cultured in HPL, little information is available on the effect of HPL on the proliferation and differentiation capacity of DPSC at a clinical scale of manufacture. We have already described a protocol for optimizing the culture conditions and seeding density of DPSC (20). Here, we expand the protocol for the large-scale production of pooled DPSC by using HPL, which reproducibility resulted in more than 3 × 108 cells in passage 2. To exclude the possibility that large-scale expansion can affect immunomodulation, the effect of up-scaled DPSC in HPL on the expression of HLA-DR was accessed. Karyotype analysis was performed to evaluate numerical and structural abnormalities, if any, in the large-scale expanded DPSC, and they were assessed for the expression of stem cells, lineage-specific oncogenes and tumor-suppressor genes. We present procedures for generating clinical quantities of human DPSC cultured in HPL and FBS with retained differentiation potential, cell-surface antigens and chromosomal stability.
Section snippets
HPL preparation
PL was prepared from 30–40 donors who had donated at the University of Malaya Blood Bank (Kuala Lumpur, Malaysia). Briefly, whole blood (WB) was collected into a quadruple blood-bag system (Baxter Health Care Corporation, Deerfield, IL, USA) and centrifuged at 4250 g for 13 min at 22°C to separate the plasma and buffy coat. Platelet-rich plasma (PRP) was prepared by mixing 4 units buffy coat (from donor group O) and 1 unit plasma (from donor group AB). Immediately after preparation, the PRP was
Proliferative response of single and pooled DPSC in HPL and FBS
Individual digestion of pulp tissues from donors (n = 9; male) yielded approximately 0.8 ± 0.2 × 106 cells/donor dental tissue. Morphologically, DPSC expanded in the presence of HPL were smaller, spindle-shaped cells and highly dense overlapping cells compared with only loosely connected cells in FBS (Figure 1A,B). The colony-forming properties of DPSC expanded in HPL and FBS were assessed. The CFU were highly packed in cells expanded in HPL compared with cells expanded in FBS (Figure 1C,D). We found
Discussion
We have demonstrated that the efficacy of DPSC cultured using HPL is highly enhanced in a large-scale set-up. The large-scale expansion of DPSC using HPL was sufficient to treat four patients, compared with FBS which accommodated up to two patients, considering a 70-kg patient needs approximately 2 × 106 MSC/kg body weight for transplantation (27), although an optimal MSC dose needs to assessed for different indications. This could certainly reduce the cost of large-scale production by 50%. Not
Acknowledgments
This work was fully funded by a University of Malaya research grant (UMRG/RG073/09HTM). Special thanks to Srijaya T. C. for reading the manuscript revision and providing valuable suggestions.
Declaration of interest: The authors report no conflicts of interest.
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