Redirection of transfusion waste and by-products for xeno-free research applications
Background and Aim: Whole blood is processed to derive a red cell concentrate, plasma and buffy coat (from which platelets can be further extracted). Unused plasma and buffy coats are common in most blood establishments and considered a liability. The redirection of these products to xeno-free applications is not complicated or time-consuming and cannot only benefit the research recipients, but also the blood establishment suppliers interested in research collaboration. The aim of this study is to describe a diverse yet by no means exhaustive list of options for preparing blood products for research applications.
Materials and Methods: Plasma and buffy coats from healthy donors were processed using basic laboratory techniques under aseptic conditions and tested for their ability to support the culture of mesenchymal stem cells in both 2D and 3D cultures using fibrin clots. The white blood cells from the buffy coats were induced by phytoheamagglutinin and CD marker expression was monitored by using qPCR.
Results: All the methods tested for preparing blood products were successful but the applicability to different settings varied greatly with the most successful being the supplementation of DMEM:F12 with 20% cryo-depleted plasma and 0.1 mg/mL platelet lysate, the formation of fibrin clots using a ratio of 3:1 (medium:plasma) and the culturing of white blood cells with 5 µg/mL phytohaemagglutinin.
Conclusions: Using the wastes and by-products of blood establishments for xeno-free cell culturing of stem cells will reduce the reliance on commercially available, ready-made products, increasing the potential for therapeutic stem cell research. Despite the benefits presented both in terms of cost and applications, further characterization and optimization of each blood product for reproducibility of results is required.
Relevance for patients: The availability of low cost xeno-free reagents will speed up therapeutic stem cell research and allow patients to receive treatments of the expected high standards at lower costs.
[1] St John AG, Zammit V. Preliminary Procedure for the Optimisation of Recovered Pooled Platelets. Austin Hematol 2019;4:1-6.
[2] Council of Europe. Good Practice Guidelines for Blood Establishments. European Directorate for the Quality of Medicines; 2018. Available from: https://www.edqm.eu/ en/good-practice-guidelines-blood-establishments. [Last accessed on 2019 Jul 05].
[3] Baron B. Biochemical Formulation of Chemically-Defined and Non-Xenogeneic Culture Media for Research and Clinical Applications of Human Stem Cells. Hyderabad: Austin Publishing Group; 2016. Available from: http:// www.austinpublishinggroup.com/ebooks. [Last accessed on 2018 Jun 20].
[4] Lubkowska A, Dolegowska B, Banfi G. Growth Factor Content in PRP and their Applicability in Medicine. J Biol Regul Homeost Agents 2012;26:3S-22S.
[5] Muraglia A, Nguyen VT, NardiniM,Mogni M, CovielloD, Dozin B, et al. Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support. Front Bioeng Biotechnol 2017;5:66.
[6] Zammit V, Baron B. Points of Good Practice for the Sampling of Cords and Culturing of Mesenchymal Stem Cells. Malta: Malta Midwives Association; 2017. Available from: https://www.um.edu.mt/library/oar/ handle/123456789/19643. [Last accessed on 2018 Jun 17].
[7] Zammit V, Brincat MR, CassarV, Muscat-BaronY, Ayers D, Baron B. MiRNA Influences in Mesenchymal Stem Cell Commitment to Neuroblast Lineage Development. Noncoding RNA Res 2018;3:232-42.
[8] Tokuda K, Baron B, Kuramitsu Y, Kitagawa T, Tokuda N, Morishige N, et al. Optimization of Fixative Solution for Retinal Morphology: A Comparison with Davidson’s Fixative and Other Fixation Solutions. Jpn J Ophthalmol 2018;62:481-90.
[9] Coligan J, Bierer B, Margulies D, Shevach E, Coico R. Basic protocol. In: Current Protocols in Immunology Immunologic Studies in Humans. New York: John Wiley and Sons, Inc.; 1991.
[10] Ledderose C, Heyn J, Limbeck E, Kreth S. Selection of Reliable Reference Genes for Quantitative Real-time PCR in Human T Cells and Neutrophils. BMC Res Notes 2011;4:427.
[11] Emam M, Thompson-Crispi K, Mallard B. The Effect of Immunological Status, in-vitro Treatment and Culture Time on Expression of Eleven Candidate Reference Genes in Bovine Blood Mononuclear Cells. BMC Immunol 2015;16:33.
[12] Radonić A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A. Guideline to Reference Gene Selection for Quantitative Real-time PCR. Biochem Biophys Res Commun 2004;313:856-62.