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Platelet lysate from whole blood-derived pooled platelet concentrates and apheresis-derived platelet concentrates for the isolation and expansion of human bone marrow mesenchymal stromal cells: production process, content and identification of active components.

Fekete N, Gadelorge M, Fürst D, Maurer C, Dausend J, Fleury-Cappellesso S, Mailänder V, Lotfi R, Ignatius A, Sensebé L, Bourin P, Schrezenmeier H, Rojewski MT - Cytotherapy (2012)

Bottom Line: Inhibition of PDGF-BB and bFGF decreased MSC proliferation by about 20% and 50%, respectively.PL from whole blood-derived pooled platelet concentrates and apheresis platelet concentrates did not differ significantly in their growth-promoting activity on MSC.PL enhances MSC proliferation and can be regarded as a safe tool for MSC expansion for clinical purposes. \in particular, PDGF-BB and bFGF are essential components for the growth-promoting effect of PL, but are not sufficient for MSC proliferation.

View Article: PubMed Central - PubMed

Affiliation: Institut für Transfusionsmedizin, Universität Ulm und Institut für Klinische Transfusionsmedizin und Immungenetik Ulm, DRK-Blutspendedienst Baden-Württemberg-Hessen, Germany.

ABSTRACT

Background aims: The clinical use of human mesenchymal stromal cells (MSC) requires ex vivo expansion in media containing supplements such as fetal bovine serum or, alternatively, human platelet lysate (PL).

Methods: Platelet concentrates were frozen, quarantine stored, thawed and sterile filtered to obtain PL. PL content and its effect on fibroblast-colony-forming unit (CFU-F) formation, MSC proliferation and large-scale expansion were studied.

Results: PL contained high levels of basic fibroblast growth factor (bFGF), soluble CD40L (sCD40L), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB/BB (PDGF-AB/BB), chemokine (C-C) ligand 5 (CCL5; RANTES) transforming growth factor-β1 (TGF-β1) and chemokine (C-X-C) ligand 1/2/3 (GRO), with low batch-to-batch variability, and most were stable for up to 14 days. Inhibition of PDGF-BB and bFGF decreased MSC proliferation by about 20% and 50%, respectively. The strongest inhibition (about 75%) was observed with a combination of anti-bFGF + anti-PDGF-BB and anti-bFGF + anti-TGF-β1 + anti-PDGF-BB. Interestingly, various combinations of recombinant PDGF-BB, bFGF and TGF-β1 were not sufficient to promote cell proliferation. PL from whole blood-derived pooled platelet concentrates and apheresis platelet concentrates did not differ significantly in their growth-promoting activity on MSC.

Conclusions: PL enhances MSC proliferation and can be regarded as a safe tool for MSC expansion for clinical purposes. \in particular, PDGF-BB and bFGF are essential components for the growth-promoting effect of PL, but are not sufficient for MSC proliferation.

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Related in: MedlinePlus

Proliferation capacity of PL-PPC and PL-APC. (A) Comparison of MSC proliferation in 10% PL-APC (n = 4) and 10% PL-PPC (n = 6). (B) MSC proliferation assays were performed by comparing different batches of PL-PPC (n = 6) and PL-APC (n = 2) as a culture supplement at concentrations ranging from 0 to 20%. Results are expressed as mean and SD values of three independent experiments. RFU, relative fluorescence units.
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fig4: Proliferation capacity of PL-PPC and PL-APC. (A) Comparison of MSC proliferation in 10% PL-APC (n = 4) and 10% PL-PPC (n = 6). (B) MSC proliferation assays were performed by comparing different batches of PL-PPC (n = 6) and PL-APC (n = 2) as a culture supplement at concentrations ranging from 0 to 20%. Results are expressed as mean and SD values of three independent experiments. RFU, relative fluorescence units.

Mentions: PL batches that were prepared from PPC either on day 2 or day 6 after donation did not differ in their growth-promoting effects on MSC. We also compared one versus two freezing cycles to lyse the platelets, and there was no significant impact of the number of freezing cycles on the stimulatory activity of PL on MSC proliferation (data not shown). No significant differences in growth-promoting activity could be observed when MSC were cultivated with 10% of either PL-PPC and PL-APC (Figure 4A). To test for different minimal thresholds of the cell-promoting efficacy of both supplements, the medium was supplemented with increasing concentrations of PL. A dose-dependent proliferation of MSC could be observed when pre-established MSC were cultured in alpha-MEM medium supplemented with heparin and 0–20% PL derived from either PPC or APC (Figure 4B). No significant difference between PL-PPC and PL-APC was observed. The optimal concentration of PL was determined as 10% as 15% and 20% of PL did not result in a significant increase in cell proliferation.


Platelet lysate from whole blood-derived pooled platelet concentrates and apheresis-derived platelet concentrates for the isolation and expansion of human bone marrow mesenchymal stromal cells: production process, content and identification of active components.

Fekete N, Gadelorge M, Fürst D, Maurer C, Dausend J, Fleury-Cappellesso S, Mailänder V, Lotfi R, Ignatius A, Sensebé L, Bourin P, Schrezenmeier H, Rojewski MT - Cytotherapy (2012)

Proliferation capacity of PL-PPC and PL-APC. (A) Comparison of MSC proliferation in 10% PL-APC (n = 4) and 10% PL-PPC (n = 6). (B) MSC proliferation assays were performed by comparing different batches of PL-PPC (n = 6) and PL-APC (n = 2) as a culture supplement at concentrations ranging from 0 to 20%. Results are expressed as mean and SD values of three independent experiments. RFU, relative fluorescence units.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3400099&req=5

fig4: Proliferation capacity of PL-PPC and PL-APC. (A) Comparison of MSC proliferation in 10% PL-APC (n = 4) and 10% PL-PPC (n = 6). (B) MSC proliferation assays were performed by comparing different batches of PL-PPC (n = 6) and PL-APC (n = 2) as a culture supplement at concentrations ranging from 0 to 20%. Results are expressed as mean and SD values of three independent experiments. RFU, relative fluorescence units.
Mentions: PL batches that were prepared from PPC either on day 2 or day 6 after donation did not differ in their growth-promoting effects on MSC. We also compared one versus two freezing cycles to lyse the platelets, and there was no significant impact of the number of freezing cycles on the stimulatory activity of PL on MSC proliferation (data not shown). No significant differences in growth-promoting activity could be observed when MSC were cultivated with 10% of either PL-PPC and PL-APC (Figure 4A). To test for different minimal thresholds of the cell-promoting efficacy of both supplements, the medium was supplemented with increasing concentrations of PL. A dose-dependent proliferation of MSC could be observed when pre-established MSC were cultured in alpha-MEM medium supplemented with heparin and 0–20% PL derived from either PPC or APC (Figure 4B). No significant difference between PL-PPC and PL-APC was observed. The optimal concentration of PL was determined as 10% as 15% and 20% of PL did not result in a significant increase in cell proliferation.

Bottom Line: Inhibition of PDGF-BB and bFGF decreased MSC proliferation by about 20% and 50%, respectively.PL from whole blood-derived pooled platelet concentrates and apheresis platelet concentrates did not differ significantly in their growth-promoting activity on MSC.PL enhances MSC proliferation and can be regarded as a safe tool for MSC expansion for clinical purposes. \in particular, PDGF-BB and bFGF are essential components for the growth-promoting effect of PL, but are not sufficient for MSC proliferation.

View Article: PubMed Central - PubMed

Affiliation: Institut für Transfusionsmedizin, Universität Ulm und Institut für Klinische Transfusionsmedizin und Immungenetik Ulm, DRK-Blutspendedienst Baden-Württemberg-Hessen, Germany.

ABSTRACT

Background aims: The clinical use of human mesenchymal stromal cells (MSC) requires ex vivo expansion in media containing supplements such as fetal bovine serum or, alternatively, human platelet lysate (PL).

Methods: Platelet concentrates were frozen, quarantine stored, thawed and sterile filtered to obtain PL. PL content and its effect on fibroblast-colony-forming unit (CFU-F) formation, MSC proliferation and large-scale expansion were studied.

Results: PL contained high levels of basic fibroblast growth factor (bFGF), soluble CD40L (sCD40L), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB/BB (PDGF-AB/BB), chemokine (C-C) ligand 5 (CCL5; RANTES) transforming growth factor-β1 (TGF-β1) and chemokine (C-X-C) ligand 1/2/3 (GRO), with low batch-to-batch variability, and most were stable for up to 14 days. Inhibition of PDGF-BB and bFGF decreased MSC proliferation by about 20% and 50%, respectively. The strongest inhibition (about 75%) was observed with a combination of anti-bFGF + anti-PDGF-BB and anti-bFGF + anti-TGF-β1 + anti-PDGF-BB. Interestingly, various combinations of recombinant PDGF-BB, bFGF and TGF-β1 were not sufficient to promote cell proliferation. PL from whole blood-derived pooled platelet concentrates and apheresis platelet concentrates did not differ significantly in their growth-promoting activity on MSC.

Conclusions: PL enhances MSC proliferation and can be regarded as a safe tool for MSC expansion for clinical purposes. \in particular, PDGF-BB and bFGF are essential components for the growth-promoting effect of PL, but are not sufficient for MSC proliferation.

Show MeSH
Related in: MedlinePlus