Standardization of platelet releasate products for clinical applications in cell therapy: A mathematical approach

Francesco Agostini, Jerry Polesel, Monica Battiston, Elisabetta Lombardi, Stefania Zanolin, Alessandro Da Ponte, Giuseppe Astori, Cristina Durante, Mario Mazzucato

Research output: Contribution to journalArticle

Abstract

Background: Standardized animal-free components are required for manufacturing cell-based medicinal products. Human platelet concentrates are sources of growth factors for cell expansion but such products are characterized by undesired variability. Pooling together single-donor products improves consistency, but the minimal pool sample size was never determined. Methods: Supernatant rich in growth factors (SRGF) derived from n = 44 single-donor platelet-apheresis was obtained by CaCl2 addition. n = 10 growth factor concentrations were measured. The data matrix was analyzed by a novel statistical algorithm programmed to create 500 groups of random data from single-donor SRGF and to repeat this task increasing group statistical sample size from n = 2 to n = 20. Thereafter, in created groups (n = 9500), the software calculated means for each growth factor and, matching groups with the same sample size, the software retrieved the percent coefficient of variation (CV) between calculated means. A 20% CV was defined as threshold. For validation, we assessed the CV of concentrations measured in n = 10 pools manufactured according to algorithm results. Finally, we compared growth rate and differentiation potential of adipose-derived stromal/stem cells (ASC) expanded by separate SRGF pools. Results: Growth factor concentrations in single-donor SRGF were characterized by high variability (mean (pg/ml)-CV); VEGF: 950-81.4; FGF-b: 27-74.6; PDGF-AA: 7883-28.8; PDGF-AB: 107834-32.5; PDGF-BB: 11142-48.4; Endostatin: 305034-16.2; Angiostatin: 197284-32.9; TGF-β1: 68382-53.7; IGF-I: 70876-38.3; EGF: 2411-30.2). In silico performed analysis suggested that pooling n = 16 single-donor SRGF reduced CV below 20%. Concentrations measured in 10 pools of n = 16 single SRGF were not different from mean values measured in single SRGF, but the CV was reduced to or below the threshold. Separate SRGF pools failed to differently affect ASC growth rate (slope pool A = 0.6; R2 = 0.99; slope pool B = 0.7; R2 0.99) or differentiation potential. Discussion: Results deriving from our algorithm and from validation utilizing real SRGF pools demonstrated that pooling n = 16 single-donor SRGF products can ameliorate variability of final growth factor concentrations. Different pools of n = 16 single donor SRGF displayed consitent capability to modulate growth and differentiation potential of expanded ASC. Increasing the pool size should not further improve product composition.

Original languageEnglish
Article number107
JournalJournal of Translational Medicine
Volume15
Issue number1
DOIs
Publication statusPublished - May 19 2017

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Cell- and Tissue-Based Therapy
Platelets
Standardization
Intercellular Signaling Peptides and Proteins
Blood Platelets
Stromal Cells
Stem cells
Sample Size
Stem Cells
Software
Growth
Angiostatins
Endostatins
Cellular manufacturing
Blood Component Removal
Cell growth
Insulin-Like Growth Factor I
Epidermal Growth Factor
Computer Simulation
Vascular Endothelial Growth Factor A

Keywords

  • ASC ex vivo expansion
  • Cell therapy
  • Good manufacturing practice
  • Growth factors from human platelets
  • Mathematical algorithm
  • Releasate standardization

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Standardization of platelet releasate products for clinical applications in cell therapy : A mathematical approach. / Agostini, Francesco; Polesel, Jerry; Battiston, Monica; Lombardi, Elisabetta; Zanolin, Stefania; Da Ponte, Alessandro; Astori, Giuseppe; Durante, Cristina; Mazzucato, Mario.

In: Journal of Translational Medicine, Vol. 15, No. 1, 107, 19.05.2017.

Research output: Contribution to journalArticle

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abstract = "Background: Standardized animal-free components are required for manufacturing cell-based medicinal products. Human platelet concentrates are sources of growth factors for cell expansion but such products are characterized by undesired variability. Pooling together single-donor products improves consistency, but the minimal pool sample size was never determined. Methods: Supernatant rich in growth factors (SRGF) derived from n = 44 single-donor platelet-apheresis was obtained by CaCl2 addition. n = 10 growth factor concentrations were measured. The data matrix was analyzed by a novel statistical algorithm programmed to create 500 groups of random data from single-donor SRGF and to repeat this task increasing group statistical sample size from n = 2 to n = 20. Thereafter, in created groups (n = 9500), the software calculated means for each growth factor and, matching groups with the same sample size, the software retrieved the percent coefficient of variation (CV) between calculated means. A 20{\%} CV was defined as threshold. For validation, we assessed the CV of concentrations measured in n = 10 pools manufactured according to algorithm results. Finally, we compared growth rate and differentiation potential of adipose-derived stromal/stem cells (ASC) expanded by separate SRGF pools. Results: Growth factor concentrations in single-donor SRGF were characterized by high variability (mean (pg/ml)-CV); VEGF: 950-81.4; FGF-b: 27-74.6; PDGF-AA: 7883-28.8; PDGF-AB: 107834-32.5; PDGF-BB: 11142-48.4; Endostatin: 305034-16.2; Angiostatin: 197284-32.9; TGF-β1: 68382-53.7; IGF-I: 70876-38.3; EGF: 2411-30.2). In silico performed analysis suggested that pooling n = 16 single-donor SRGF reduced CV below 20{\%}. Concentrations measured in 10 pools of n = 16 single SRGF were not different from mean values measured in single SRGF, but the CV was reduced to or below the threshold. Separate SRGF pools failed to differently affect ASC growth rate (slope pool A = 0.6; R2 = 0.99; slope pool B = 0.7; R2 0.99) or differentiation potential. Discussion: Results deriving from our algorithm and from validation utilizing real SRGF pools demonstrated that pooling n = 16 single-donor SRGF products can ameliorate variability of final growth factor concentrations. Different pools of n = 16 single donor SRGF displayed consitent capability to modulate growth and differentiation potential of expanded ASC. Increasing the pool size should not further improve product composition.",
keywords = "ASC ex vivo expansion, Cell therapy, Good manufacturing practice, Growth factors from human platelets, Mathematical algorithm, Releasate standardization",
author = "Francesco Agostini and Jerry Polesel and Monica Battiston and Elisabetta Lombardi and Stefania Zanolin and {Da Ponte}, Alessandro and Giuseppe Astori and Cristina Durante and Mario Mazzucato",
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T1 - Standardization of platelet releasate products for clinical applications in cell therapy

T2 - A mathematical approach

AU - Agostini, Francesco

AU - Polesel, Jerry

AU - Battiston, Monica

AU - Lombardi, Elisabetta

AU - Zanolin, Stefania

AU - Da Ponte, Alessandro

AU - Astori, Giuseppe

AU - Durante, Cristina

AU - Mazzucato, Mario

PY - 2017/5/19

Y1 - 2017/5/19

N2 - Background: Standardized animal-free components are required for manufacturing cell-based medicinal products. Human platelet concentrates are sources of growth factors for cell expansion but such products are characterized by undesired variability. Pooling together single-donor products improves consistency, but the minimal pool sample size was never determined. Methods: Supernatant rich in growth factors (SRGF) derived from n = 44 single-donor platelet-apheresis was obtained by CaCl2 addition. n = 10 growth factor concentrations were measured. The data matrix was analyzed by a novel statistical algorithm programmed to create 500 groups of random data from single-donor SRGF and to repeat this task increasing group statistical sample size from n = 2 to n = 20. Thereafter, in created groups (n = 9500), the software calculated means for each growth factor and, matching groups with the same sample size, the software retrieved the percent coefficient of variation (CV) between calculated means. A 20% CV was defined as threshold. For validation, we assessed the CV of concentrations measured in n = 10 pools manufactured according to algorithm results. Finally, we compared growth rate and differentiation potential of adipose-derived stromal/stem cells (ASC) expanded by separate SRGF pools. Results: Growth factor concentrations in single-donor SRGF were characterized by high variability (mean (pg/ml)-CV); VEGF: 950-81.4; FGF-b: 27-74.6; PDGF-AA: 7883-28.8; PDGF-AB: 107834-32.5; PDGF-BB: 11142-48.4; Endostatin: 305034-16.2; Angiostatin: 197284-32.9; TGF-β1: 68382-53.7; IGF-I: 70876-38.3; EGF: 2411-30.2). In silico performed analysis suggested that pooling n = 16 single-donor SRGF reduced CV below 20%. Concentrations measured in 10 pools of n = 16 single SRGF were not different from mean values measured in single SRGF, but the CV was reduced to or below the threshold. Separate SRGF pools failed to differently affect ASC growth rate (slope pool A = 0.6; R2 = 0.99; slope pool B = 0.7; R2 0.99) or differentiation potential. Discussion: Results deriving from our algorithm and from validation utilizing real SRGF pools demonstrated that pooling n = 16 single-donor SRGF products can ameliorate variability of final growth factor concentrations. Different pools of n = 16 single donor SRGF displayed consitent capability to modulate growth and differentiation potential of expanded ASC. Increasing the pool size should not further improve product composition.

AB - Background: Standardized animal-free components are required for manufacturing cell-based medicinal products. Human platelet concentrates are sources of growth factors for cell expansion but such products are characterized by undesired variability. Pooling together single-donor products improves consistency, but the minimal pool sample size was never determined. Methods: Supernatant rich in growth factors (SRGF) derived from n = 44 single-donor platelet-apheresis was obtained by CaCl2 addition. n = 10 growth factor concentrations were measured. The data matrix was analyzed by a novel statistical algorithm programmed to create 500 groups of random data from single-donor SRGF and to repeat this task increasing group statistical sample size from n = 2 to n = 20. Thereafter, in created groups (n = 9500), the software calculated means for each growth factor and, matching groups with the same sample size, the software retrieved the percent coefficient of variation (CV) between calculated means. A 20% CV was defined as threshold. For validation, we assessed the CV of concentrations measured in n = 10 pools manufactured according to algorithm results. Finally, we compared growth rate and differentiation potential of adipose-derived stromal/stem cells (ASC) expanded by separate SRGF pools. Results: Growth factor concentrations in single-donor SRGF were characterized by high variability (mean (pg/ml)-CV); VEGF: 950-81.4; FGF-b: 27-74.6; PDGF-AA: 7883-28.8; PDGF-AB: 107834-32.5; PDGF-BB: 11142-48.4; Endostatin: 305034-16.2; Angiostatin: 197284-32.9; TGF-β1: 68382-53.7; IGF-I: 70876-38.3; EGF: 2411-30.2). In silico performed analysis suggested that pooling n = 16 single-donor SRGF reduced CV below 20%. Concentrations measured in 10 pools of n = 16 single SRGF were not different from mean values measured in single SRGF, but the CV was reduced to or below the threshold. Separate SRGF pools failed to differently affect ASC growth rate (slope pool A = 0.6; R2 = 0.99; slope pool B = 0.7; R2 0.99) or differentiation potential. Discussion: Results deriving from our algorithm and from validation utilizing real SRGF pools demonstrated that pooling n = 16 single-donor SRGF products can ameliorate variability of final growth factor concentrations. Different pools of n = 16 single donor SRGF displayed consitent capability to modulate growth and differentiation potential of expanded ASC. Increasing the pool size should not further improve product composition.

KW - ASC ex vivo expansion

KW - Cell therapy

KW - Good manufacturing practice

KW - Growth factors from human platelets

KW - Mathematical algorithm

KW - Releasate standardization

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