Received: September 19, 2018
Accepted: November 8, 2018
Mediterr J Hematol Infect Dis 2019, 11(1): e2019021 DOI 10.4084/MJHID.2019.021
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Platelet-rich plasma (PRP) is an inexpensive and safe substitute of recombinant growth factors in vitro and in vivo.
Due to its putative effect on tissue repair, the use of autologous PRP
has become largely popular. Recently, a jellified PRP derivative
obtained from umbilical cord blood (CB) has been utilized in vivo
to treat mucosal and cutaneous lesions. Nevertheless, whether PRP
derived from CB and adult blood display different potency in promoting
cell growth in vitro has been
rarely investigated. In this study, we compared cytokine profile and
mesenchymal cell growth supporting the ability of platelet lysate
obtained from adult and cord blood. Our in vitro results strongly back the utilization of CB platelet lysate in vivo,
as an efficacious, safe and inexpensive alternative to promote damaged
tissue healing when the autologous PRP is contraindicated. Moreover,
the policy of manufacturing CB platelet lysate can limit the current
disposal of many collected CB units not suitable for transplant due to
their low nucleated cell count.
It is widely acknowledged that cord blood contains a high amount of growth factors. Nevertheless, there are very few studies investigating whether cord and adult PLs display different potency in promoting cell growth in vitro. Similarly, the types of cytokines and growth factors released by fetal platelets have been only partially investigated.[14,15]
Materials and Methods
Mesenchymal cell cultures. Mesenchymal cells (MSCs) were obtained from bone marrow (BM) samples of a healthy BM donor using residual nucleated cells recovered from filters utilized for the BM graft filtration, after repeated washed with PBS. After a density gradient centrifugation (Lympholyte-H, Cedarlane Lab, Canada), mononuclear cells were then collected and seeded at 10x106 /ml in αMEM (Lonza, Italy) containing 20% FBS (Life Technologies, USA). After three days, non-adherent cells were discharged, and adherent cells were left to grow until confluence. Cultures were maintained at 37°C in humidified 5% CO2 atmosphere, and the medium was changed twice a week. When 80% of confluence was achieved, MSCs were detached by Trypsin 1mM EDTA (Sigma Aldrich, Italy), counted and sub-cultured in the same medium containing 10% FBS. The cell population doubling (PD) rate was calculated in MSC cultures after the passage 3 as previously reported, according to the formula PD = log10(N)/log10(2), where N was the ratio between harvested and seeded cells. The colony forming unit-fibroblast (CFU-F) assay was performed by seeding MSCs (from passage 5 or higher), in 60 mm dishes at a concentration of 1-2 cell/cm2 in αMEM and 20% FBS. After two weeks of culture, colonies were counted, fixed and visualized by crystal violet staining. In order to compare the effect of CB and adult blood PLs, FBS was substituted by 20% of PL obtained either pooling the eight samples of CB PLs or the four samples of adult blood PLs.
Cytokine assay. The cytokine and growth factors profile were evaluated in adult and CB starting plasma samples and in final adult and CB PL products. The Bio-Plex Human Cytokine 27-Plex Assay and the Bio-Plex MAGPIX™ Multiplex Reader (Bio-Rad, CA, USA) were utilized. The following molecules were dosed: growth factors (G-CSF, GM-CSF, VEGF, FGF, PDGF), chemokines (MCP-1/CCL2, MIP-1a/CCL3, MIP-1b/CCL4, RANTES/CCL5, Eotaxin/CCL11, IP-10/CXCL10), interleukins (IL-1β, IL-1RA, IL-2,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL10, IL-12, IL-13,IL-15, IL-17), and pro-inflammatory mediators (TNF-α, INF-γ). All samples were evaluated in duplicate, according to the manufacturer’s instructions.
Statistics. Continuous variables were expressed as mean values + standard error (SEM), and they were analyzed through the Mann-Whitney U test or the Kruskal-Wallis test. The GraphPad PRISM 5.0 software and the IBM SPSS Statistics 21.0 software were utilized. The p-value <0.05 were regarded as statistically significant.
Results and Discussion
|Figure 1. Mean values + SEM of cytokine levels in plasma and platelet lysates obtained from cord blood (yellow columns) and adult blood (red columns). (A) Inflammatory cytokines (B) Anti-inflammatory cytokines. * = p < 0.05; ** = p <0.01.|
|Figure 2. Mean values + SEM of cytokine levels in plasma and platelet lysates obtained from cord blood (yellow columns) and adult blood (red columns). (A) Chemokines (B) Growth factors * = p < 0.05; ** = p <0.01.|
Cord platelet lysates more efficiently promote cell proliferation than their adult blood counterparts. The kinetic of MSC expansion in cultures supplemented with FBS, adult PLs or CB PLs is shown in Figure 3A. As shown by the cumulative PD of three single experiments, CB PLs (obtained by mixing 8 single PLs samples in 3 different pools) were significantly more active in supporting MSC growth over the whole time of culture (p < 0.001 at every passage). Similarly, fibroblast colony yield in the presence of CB PLs was significantly higher than in culture supplemented with FBS or adult PLs (p = 0.003; Figure 3 B). On the whole, our data are in partial agreement with those reported by Parazzi et al. in adipose tissue-derived MSCs. In fact, the authors found no difference between adult and cord blood platelet releasate in supporting cell proliferation, whereas both were much more active than FBS. The use of MSCs from different sources (BM in our study and adipose tissue in Parazzi’ study), in addition to the use of different PRP derivatives (platelet releasate and platelet lysate), may account for these differences.
In conclusion, our data strongly support the exploitation of cord blood PLs in vitro and prompt its utilization in vivo, to promote damaged tissue healing. Notably, this could be a more efficacious and safer alternative to the autologous PRP. Indeed, cord blood platelet lysate could be a valid alternative in case of contraindication to the autologous PRP for patient refusal or discomfort, unsuitability of venous accesses, or coexistence of inflammatory, autoimmune or hematological diseases. In the meanwhile, the manufacture of CB derivative as medical products for local use, could limit the current disposal of several CB units collected for solidary donation, and not suitable for transplant due to their low nucleated cell count.
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