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Original Article

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Blood Res 2020; 55(1):

Published online March 31, 2020

https://doi.org/10.5045/br.2020.55.1.35

© The Korean Society of Hematology

Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

Hoda E. Mohamed1, Mervat E. Asker1, Nahla S. Kotb2, Akram M. El Habab2

Author Info. +

1Department of Biochemistry, Faculty of Pharmacy, Zagazig University, 2National Organization for Research and Control of Biologicals, Giza, Egypt

Correspondence to : Akram M. El Habab, B.S.
National Organization for Research and Control of Biologicals, Giza, Agouza 12654, Egypt
E-mail: akram.elhabab@yahoo.com

Received: October 7, 2019; Revised: November 27, 2019; Accepted: February 21, 2020

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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Background
Fetal bovine serum (FBS) has been used to support the growth and proliferation of mammalian cells for decades. Owing to several risk factors associated with FBS, several trials have been conducted to evaluate substitutes to FBS with the same efficiency and the lower risk issues.
Methods
In this study, human platelet lysate (HPL) derived from activated human platelets was evaluated as an alternative to FBS due to the associated risk factors. To evaluate the efficiency of the preparation process, platelet count was performed before and after activation. The concentrations of several growth factors and proteins were measured to investigate HPL efficiency. HPL stability was studied at regular intervals, and optimal heparin concentration required to prevent gel formation in various media was determined. The biological activity of HPL and FBS was compared by evaluating the growth performance of Vero and Hep-2 cell lines.
Results
Result of platelet count assay revealed the efficiency of HPL preparation process. Growth factor concentrations in HPL were significantly higher than those in FBS, while the protein content of HPL was lower than that of FBS. Stability study data showed that the prepared HPL was stable for up to 15 months at -20°C. Ideal heparin concentration to be used in different media was dependent on calcium concentration. Results of cell viability assay showed that HPL was superior to FBS in supporting the growth and proliferation of Vero and Hep-2 cells.
Conclusion
The HPL prepared by the mechanical activation of platelets may serve as an efficient alternative to FBS in cell culture process.

Keywords Vero cell line, Hep-2 cell line, Heparin, Human platelet lysate, Growth factors, Viability assay

Article

Original Article

Blood Res 2020; 55(1): 35-43

Published online March 31, 2020 https://doi.org/10.5045/br.2020.55.1.35

Copyright © The Korean Society of Hematology.

Human platelet lysate efficiency, stability, and optimal heparin concentration required in culture of mammalian cells

Hoda E. Mohamed1, Mervat E. Asker1, Nahla S. Kotb2, Akram M. El Habab2

1Department of Biochemistry, Faculty of Pharmacy, Zagazig University, 2National Organization for Research and Control of Biologicals, Giza, Egypt

Correspondence to:Akram M. El Habab, B.S.
National Organization for Research and Control of Biologicals, Giza, Agouza 12654, Egypt
E-mail: akram.elhabab@yahoo.com

Received: October 7, 2019; Revised: November 27, 2019; Accepted: February 21, 2020

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background
Fetal bovine serum (FBS) has been used to support the growth and proliferation of mammalian cells for decades. Owing to several risk factors associated with FBS, several trials have been conducted to evaluate substitutes to FBS with the same efficiency and the lower risk issues.
Methods
In this study, human platelet lysate (HPL) derived from activated human platelets was evaluated as an alternative to FBS due to the associated risk factors. To evaluate the efficiency of the preparation process, platelet count was performed before and after activation. The concentrations of several growth factors and proteins were measured to investigate HPL efficiency. HPL stability was studied at regular intervals, and optimal heparin concentration required to prevent gel formation in various media was determined. The biological activity of HPL and FBS was compared by evaluating the growth performance of Vero and Hep-2 cell lines.
Results
Result of platelet count assay revealed the efficiency of HPL preparation process. Growth factor concentrations in HPL were significantly higher than those in FBS, while the protein content of HPL was lower than that of FBS. Stability study data showed that the prepared HPL was stable for up to 15 months at -20°C. Ideal heparin concentration to be used in different media was dependent on calcium concentration. Results of cell viability assay showed that HPL was superior to FBS in supporting the growth and proliferation of Vero and Hep-2 cells.
Conclusion
The HPL prepared by the mechanical activation of platelets may serve as an efficient alternative to FBS in cell culture process.

Keywords: Vero cell line, Hep-2 cell line, Heparin, Human platelet lysate, Growth factors, Viability assay

Fig 1.

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Figure 1.Preparation of human platelet lysate from PRP.
Blood Research 2020; 55: 35-43https://doi.org/10.5045/br.2020.55.1.35

Fig 2.

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Figure 2.Growth factor contents in FBS and HPL samples. IGF-1 (A), TGF-β (B), VEGF (C), EGF (D), FGF (E), PDGF (F). Values are expressed as means±SD (N=3).
a)Significantly different from FBS samples at P <0.05.
Blood Research 2020; 55: 35-43https://doi.org/10.5045/br.2020.55.1.35

Fig 3.

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Figure 3.Growth factor stability in HPL and FBS samples stored over 15 months. VEGF (A), IGF-1 (B), TGF-β (C). Values are expressed as means±SD (N=3).
Blood Research 2020; 55: 35-43https://doi.org/10.5045/br.2020.55.1.35

Fig 4.

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Figure 4.(A) MTT reduction curve for Vero cells cultured in the presence of 10% FBS, 10% HPL, and 5% HPL and in SFM. Data are expressed as means of three independent experiments (P <0.05). (B) MTT reduction difference between cells cultivated in 10% and 5% HPL and SFM and those cultured in 10% FBS (P <0.05).
Blood Research 2020; 55: 35-43https://doi.org/10.5045/br.2020.55.1.35

Fig 5.

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Figure 5.(A) MTT reduction curve for Hep-2 cells cultured in the presence of 10% FBS, 10% HPL, and 5% HPL and in SFM (P <0.05). Data are expressed as means±SD of three independent experiments. (B) MTT reduction difference between cells cultivated in 10% and 5% HPL and SFM and those cultured in 10% FBS (P <0.05).
Blood Research 2020; 55: 35-43https://doi.org/10.5045/br.2020.55.1.35

Fig 6.

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Figure 6.Count of Vero cells cultured in 10% FBS and 10% HPL. Data are expressed as means±SD of three independent experiments (P >0.05).
Blood Research 2020; 55: 35-43https://doi.org/10.5045/br.2020.55.1.35

Fig 7.

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Figure 7.Count of Hep-2 cells cultivated in 10% FBS and 10% HPL. Data are expressed as means±SD of three independent experiments (P > 0.05).
Blood Research 2020; 55: 35-43https://doi.org/10.5045/br.2020.55.1.35

Different concentrations of heparin in cell culture media at different incubation conditions..

Abbreviations: (+), coagulation; (−), no coagulation; RT, room temperature; temp, incubation temperature..


Different concentrations of calcium in deionized water containing 10% HPL at different incubation conditions..

Abbreviations: (+), coagulation; (−), no coagulation; mM, millimolar; RT, room temperature; temp, incubation temperature..


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