Background and Overview
Mesenchymal stromal cells (MSCs) are adult multipotent cells that can be isolated from several human tissues. MSCs represent a novel and attractive tool in strategies of cellular therapy. For in vivo use, MSCs have to be ex vivo expanded in order to reach the numbers suitable for their clinical applications (e.g., MultiPL’e sclerosis). Despite being efficacious, the use of fetal bovine serum (FBS; also known as fetal calf serum) for MSC ex vivo expansion for clinical purposes raises concerns related to immunization and transmission of zoonoses; the standardization of expansion methods, possibly devoid of animal components, such as those based on human MultiPL’, are discussed in this report (Bernardo et al, 2011).
In addition, these methods are not appropriate to meet the expected future demand for quality-assured MSCs for human therapeutic use. Hence, it is imperative to develop an effective MSC production system, which should be controllable, reproducible, and scalable. To this end, efforts have been made by several international research groups to develop (i) alternative media either by replacing FBS with human-sourced supplements (such as human serum – huS – or MultiPL’) or by identifying defined serum-free formulations consisting of key growth/attachment factors, and (ii) controlled bioreactor protocols (Jung et al, 2012).
Platelet Lysate (MultiPL’) contains a cocktail of growth factors and cytokines, which actively participates in tissue repair and its clinical application has been broadly described (Ruggiu et al, 2013).
Due to the limited frequency in most tissue sources, ex vivo expansion of MSC is required in a manner which is compliant with good manufacturing practice (GMP) guidelines to yield clinically relevant cell doses. Most manufacturing protocols still use FBS as the cell culture supplement to isolate and to expand MSC. However, the high lot-to-lot variability as well as risk of contamination and immunization call for xenogenic-free culture conditions can be problematic. In terms of standardization, chemically defined media appear as the ultimate achievement. Since these media need to maintain all key cellular and therapy-relevant features of MSC, the development of chemically defined media is still – albeit highly investigated – only in its beginning. The current alternatives to FBS rely on human blood-derived components: plasma, serum, umbilical cord blood (UCB) serum, and platelet derivatives like MultiPL’ (Bieback et al, 2013; Kinzebach et al, 2013).
In view of the above, within the past years, many laboratories have adapted their culture conditions from FBS to MultiPL’, which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for FBS are clearly to be preferred. MultiPL’ is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in MultiPL’ such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of MultiPL’ necessitate addition of anticoagulants such as heparins to prevent gelatinization of MultiPL’ medium, and their concentration must be standardised. Labels for description of MultiPL’-such as “xenogen-free,” “animal-free” and “serum free”-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of MultiPL’ has several advantages and disadvantages : they must be taken into account because the choice of cell culture additive has a impact on cell preparations (Hemeda et al, 2014).
Rubio-Azpeitia et al (2014) aimed to identify current in vitro research exploring platelet-rich plasma (PRP; used as MultiPL’ – MultiPL’ – or releasate) effects in human Mesenchymal Stem Cells (MSCs) that may encourage or limit the clinical application of MSCs along with PRP. After a systematic search, they identified 57 in vitro studies, focused on optimization of MSC manufacturing, and expanding knowledge about how PRP modifies MSCs behaviour for translational purposes. Influences of PRP on proliferation, migration, stemness, preservation of MSC immune-modulatory properties and appearance of senescence phenotype were explored. Overall PRP stimulates MSC proliferation, preserves MSCs multipotency and does not interfere with any lineage differentiation. PRP (as MultiPL’ or releasate) preserves the immune-privileged potential of MSCs and may delay the appearance of the senescent phenotype. Currently there are few data linking precise molecules and biological mechanisms. Various gaps of knowledge need to be addressed in order to obtain enough useful information for translational purposes (Rubio-Azpeitia et al, 2014).
Most clinical applications of human multipotent mesenchymal stromal cells (MSCs) for cell therapy, tissue engineering, regenerative medicine, and treatment of immune and inflammatory diseases require a phase of isolation and ex vivo expansion allowing a clinically meaningful cell number to be reached. Conditions used for cell isolation and expansion should meet strict quality and safety requirements. This is particularly true for the growth medium used for MSC isolation and expansion. Basal growth media used for MSC expansion are supplemented with MultiPL’ nutrients and growth factors. FBS has long been the gold standard medium supplement for laboratory-scale MSC culture. However, FBS has a poorly characterized composition and poses risk factors, as it may be a source of xenogenic antigens and zoonotic infections. FBS has therefore become undesirable as a growth medium supplement for isolating and expanding MSCs for human therapy protocols. In recent years, human blood materials, and most particularly lysates and releasates of platelet concentrates have emerged as efficient medium supplements for isolating and expanding MSCs from various origins. A review by Shih et al (2014) analyzes the advantages and limits of using human platelet materials as medium supplements for MSC isolation and expansion. The authors present the modes of production of allogeneic and autologous platelet concentrates, measures taken to ensure optimal pathogen safety profiles, and methods of preparing MultiPL’s for MSC expansion. They also discuss the supply of such blood preparations. Produced under optimal conditions of standardization and safety, human platelet materials can become the future ‘gold standard’ supplement for ex vivo production of MSCs for translational medicine and cell therapy applications (Shih et al, 2014).
The following is a summary of the individual studies in this area, in respect of both MSCs and other cell types, identified in a PUBMED search in September 2014.
List of References :
- Bernardo ME, Cometa AM, Pagliara D, Vinti L, Rossi F, Cristantielli R, Palumbo G, Locatelli F. ex vivo expansion of mesenchymal stromal cells. Best Pract Res Clin Haematol. 2011 Mar;24(1):73-81. doi: 10.1016/j.beha.2010.11.002. Epub 2011 Feb 23. Review. PubMed PMID: 21396595.
- Bieback K, Hecker A, Kocaömer A, Lannert H, Schallmoser K, Strunk D, Klüter H. Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow. Stem Cells. 2009 Sep; 27(9): 2331-41. doi: 10.1002/stem.139. PubMed PMID: 19544413.
- Hemeda H, Giebel B, Wagner W. Evaluation of human hPL versus fetal bovine serum for culture of mesenchymal stromal cells. Cytotherapy. 2014 Feb;16(2):170-80. doi: 10.1016/j.jcyt.2013.11.004. PubMed PMID: 24438898.
- Jung S, Panchalingam KM, Wuerth RD, Rosenberg L, Behie LA. Large-scale production of human mesenchymal stem cells for clinical applications. Biotechnol Appl Biochem. 2012 Mar-Apr; 59(2): 106-20. doi: 10.1002/bab.1006. Review. PubMed PMID: 23586791.
- Kinzebach S, Bieback K. Expansion of Mesenchymal Stem/Stromal cells under xenogenic-free culture conditions. Adv Biochem Eng Biotechnol. 2013;129:33-57. doi: 10.1007/10_2012_134. Review. PubMed PMID: 22777242.
- Rubio-Azpeitia E, Andia I. Partnership between platelet-rich plasma and mesenchymal stem cells: in vitro experience. Muscles Ligaments Tendons J. 2014 May 8;4(1):52-62. eCollection 2014 Jan. Review. PubMed PMID: 24932448; PubMed Central PMCID: PMC4049651.
- Ruggiu A, Ulivi V, Sanguineti F, Cancedda R, Descalzi F. The effect of Platelet Lysate on osteoblast proliferation associated with a transient increase of the inflammatory response in bone regeneration. Biomaterials. 2013 Dec;34(37):9318-30. doi: 10.1016/j.biomaterials.2013.08.018. Epub 2013 Sep 5. PubMed PMID: 24012435.
- Shih DT, Burnouf T. Preparation, quality criteria, and properties of human blood hPL supplements forex vivo stem cell expansion. N Biotechnol. 2014 Jun 11. pii: S1871-6784(14)00672-4. doi: 10.1016/j.nbt.2014.06.001. [Epub ahead of print] PubMed PMID: 24929129.
- Trébéden-Negre H, Vieillard V, Rosenzwajg M, Garderet L, Cherai M, Nguyen-Quoc S, Tanguy ML, Norol F. Polyvalent immunoglobulins, platelet lysate and lenalidomide: cocktail for polyfunctional NK cells expansion for multiple myeloma. 2016 Dec 12. doi: 10.1038/bmt.2016.311. PubMed PMID:27941770.
- Viau S, Chabrand L, Eap S, Lorant J, Rouger K, Goudaliez F, Sumian C, Delorme B. Pathogen reduction through additive-free short-wave UV light irradiation retains the optimal efficacy of human platelet lysate for the expansion of human bone marrow mesenchymal stem cells. 2017 Aug 1. doi: 10.1371/journal.pone.0181406. PubMed PMID:28763452.
- Saury C, Lardenois A, Schleder C, Leroux I, Lieubeau B, David L, Charrier M, Guével L, Viau S, Delorme B, Rouger K. Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches. Stem Cell Research & Therapy, 9 (2018); PubMed PMID 29720259.