Human platelet lysate permits scale-up of dental pulp stromal cells for clinical applications

Abstract

BACKGROUND AIMS. Dental pulp stromal cells (DPSC) are considered to be a promising source of stem cells in the field of regenerative therapy. However, the usage of DPSC in transplantation requires large-scale expansion to cater for the need for clinical quantity without compromising current good manufacturing practice (cGMP). Existing protocols for cell culturing make use of fetal bovine serum (FBS) as a nutritional supplement. Unfortunately, FBS is an undesirable additive to cells because it carries the risk of transmitting viral and prion diseases. Therefore, the present study was undertaken to examine the efficacy of human platelet lysate (HPL) as a substitute for FBS in a large-scale set-up. METHODS. We expanded the DPSC in Dulbecco's modified Eagle's medium-knock-out (DMEM-KO) with either 10% FBS or 10% HPL, and studied the characteristics of DPSC at pre- (T25 culture flask) and post- (5-STACK chamber) large-scale expansion in terms of their identity, quality, functionality, molecular signatures and cytogenetic stability. RESULTS. In both pre- and post-large-scale expansion, DPSC expanded in HPL showed extensive proliferation of cells (c. 2-fold) compared with FBS; the purity, immune phenotype, colony-forming unit potential and differentiation were comparable. Furthermore, to understand the gene expression profiling, the transcriptomes and cytogenetics of DPSC expanded under HPL and FBS were compared, revealing similar expression profiles. CONCLUSIONS. We present a highly economized expansion of DPSC in HPL, yielding double the amount of cells while retaining their basic characteristics during a shorter time period under cGMP conditions, making it suitable for therapeutic applications.

Human Plasma and Human Platelet-rich Plasma as a Substitute for Fetal Calf Serum during Long-term Cultivation of Mesenchymal Dental Pulp Stem Cells

“…The hMSC cultured in media of different compositions resulted in similar morphological variances as previously described for hMSC (38,42,19,20,21). The fastest colony formation and proliferation was observed in the media containing 10% platelet rich plasma.…”

“…We are fully aware that DPSC were cultivated in a medium containing 2% FCS during first three passages and this may affect the results of the experiment, but on the other hand through this procedure we have been able to clearly define and demonstrate the changes in phenotype, proliferation ability and viability of the tested DPSC. To investigate the differences among the properties of hMSC cultivated in various media supplemented with FCS, or human blood components (49,50,51,52,19,20,21); however neither of them provides statistically relevant results on which we could build.…”

“…As the cultivation supplement we can used manufactory produced approved AB-serum or umbilical cord blood, which is very hard to obtain. Stojkovic proved that embryonic stem cells cultivated in medium with human serum retain all the characteristics of embryonic cells during long-term cultivation and have unlimited proliferation capacity while maintaining a stable karyotype (18) and Bo Chen, Ui-Lyong Lee and Vijayendram (19,20,21) proved that DPSC can be cultivated in presence of PRP without losing their SC characteristics and differentiation capacity. The effect of platelet rich plasma (PRP) and plasma (HP) derived from human blood is based on degranulation α granules from activated platelets, which contain growth factors.…”

“…Platelet concentrates are defined as components which have 3 to 5 fold higher concentration of platelets in the plasma compared to whole blood (25). It was proved that stem cells cultivated in a media enriched with PRP maintain differentiation capacity, demonstrate higher proliferative activity (19)(20)(21)(26)(27)(28)(29)(30), while it was also shown that human serum alone does not fully support the growth of human mesenchymal stem cells in vitro (31). Another option for replacing the FCS is artificially mixed supplement, but due to the complexity and not completely known composition of FCS it will be very expensive and hard to obtain it.…”

Approaches for Stimulating Proliferation of Stem Cells in Vitro

“…Tavakolinejad et al [36] have also found that treatment with hPRP resulted in a statistically significant increase in cell proliferation of human adipose-derived stem cells. In addition to human adipose-derived stem cells, increased proliferation rate for other types of stem cells with hPRP as the cell culture supplement was confirmed [37][38][39][40]. And the fastest proliferation was observed when hPRP was supplemented in a 10% concentration.…”