- Open Access
Scalable efficient expansion of mesenchymal stem cells in xeno free media using commercially available reagents
© Riordan et al. 2015
- Received: 14 April 2015
- Accepted: 3 June 2015
- Published: 17 July 2015
The rapid clinical translation of mesenchymal stem cells (MSC) has resulted in the development of cell-based strategies for multiple indications. Unfortunately one major barrier to widespread implementation of MSC-based therapies is the limited supply of fetal calf serum (FCS) used to expand cells to therapeutic numbers. Additionally, the xenogeneic element of fetal calf serum has been previously demonstrated to stimulate antibody mediated reactions and in some cases sensitization leading to anaphylaxis.
XcytePLUS™ media, a human platelet lysate based product, was used to supplement the culture medium at 5, 7.5 and 10% and compared to fetal calf serum at 10%, for human umbilical cord MSC expansion. Properties of the expanded cells were investigated.
This study demonstrated equivalent or superior effects of human platelet lysate compared to standard FCS supplemented media, based on doubling rate, without loss of identity or function, as demonstrated with flow cytometry characterization. Differentiation into osteocytes, adipocytes and chondrocytes was comparable from cells expanded in either media supplement.
These data support the implementation of human platelet lysate supplemented media as an alternative to xenogeneic containing preparations which may lead to safer MSC products with therapeutic uses.
- Mesenchymal Stem Cell
- Fetal Calf Serum
- Graft Versus Host Disease
- Bone Marrow Mesenchymal Stem Cell
- Platelet Lysate
Tissue culture originated with the work of Alexis Carrell at the beginning of the 20th century utilizing a variety of undefined media additives such as embryonic and muscle extracts mixed with saline plasma, which were able to maintain cardiac cells in vitro and allow for contractile function . The introduction of fetal calf serum (FCS) as a media additive revolutionized the field of tissue culture and allowed for laboratories to maintain stable cell lines in vitro, which catalyzed major efforts in cancer research . The concept of utilizing cells as therapeutics originated in the bone marrow transplant area, in which uncultured donor bone marrow stem cells were used for hematopoietic support to allow for high dose chemotherapy and radiation therapy . The utilization of cellular immunotherapy, in the 1980s was the first major clinical experiments that required expansion of cells in vitro. In these early experiments, it was demonstrated that fetal calf serum posed some potential to induce adverse effects. For example, a study reported that syngeneic lymphocytes cultured in FCS and subsequently administered to HIV patients resulted in Arthus reaction and fever in recipients, which was associated with antibodies to FCS components . Other studies have shown that both pre-existing natural, as well as inducible antibodies are present in patients that recognize various components of FCS [5–8]. Additionally, activation of both T cell and NK cell responses have been reported subsequent to stimulation with FCS [9, 10].
Mesenchymal stem cells (MSC) are fibroblast-shaped cells capable of multigenic differentiation into bone, fat and cartilage, as well as differentiation into neuronal, hepatic and pancreatic tissue . A number of clinical trials have been performed utilizing MSC in conditions ranging from hepatic failure , type 1 diabetes , and stroke . The majority of these clinical trials have utilized MSCs that are grown in FCS containing media. Overall safety has been demonstrated of MSC, as reported in a meta-analysis of clinical trials containing over 1,000 patients . Interestingly, in these trials administration doses were 1–3 injections, thus substantially decreasing the possibility of sensitization. Despite this, some evidence of sensitization has been reported. Specifically, Le Blanc et al. demonstrated in 12 patients being administered bone marrow MSC for treatment of steroid resistant graft versus host disease (GVHD) the development of antibodies to fetal calf serum proteins as detected by ex vivo treatment of fetal calf proteins .
In order to overcome limitations associated with FCS, numerous groups have developed serum-free tissue culture media compositions for growing and expansion of MSC. Platelet derived growth factor (PDGF) is a potent cellular mitogen and has been reported to be a significant component of FCS allowing for cellular proliferation in vitro . Since platelet lysate contains significant concentrations of PDGF, as well as numerous other mitogenic factors , investigators have utilized this as a potential substitute for FCS. In 2006 Müller et al. reported that bone marrow MSC cultures can be initiated and maintained in media in which FCS was substituted with platelet lysate . The MSC grown in platelet lysate substituted media retained both differentiation, as well as immune modulatory activity. Numerous other studies have demonstrated that various platelet lysate preparations are capable of maintaining or increasing proliferation of bone marrow MSC as a substitute for FCS [20–35]. Another report on 213 patients treated with autologous BM MSC cultured in platelet lysate reported no adverse reactions subsequent to intradisc and intrajoint administration . Some characterization has occurred of active components of platelet lysate. Specifically, antibody blocking studies have shown that up to 75% inhibition of MSC proliferation in response to platelet lysate was achieved 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, implying some components still remain unknown .
Despite advancements that have been reported utilizing platelet lysate, several obstacles still remain for large-scale commercial application, namely: (a) establishing the role that platelet lysate plays in increasing anti-inflammatory activity of MSC when compared to culture in FCS [38, 39]; (b) significant variation in MSC cultures depending on platelet donor profiles , with age being a contributing factor ; and (c) institution-specific proprietary methods for large-scale commercial production of platelet lysate, and the roles of factors such as heparin , or fibrinogen , concentration post large-scale production. Addressing these points will allow for a better standardized method for large-scale commercial production.
Here we present data using a commercially available platelet-lysate based media, XcytePLUS™, to culture human umbilical cord’s Wharton’s Jelly MSC (WJ-MSC) in comparison to media containing FCS. Additionally, the culture system described is completely free of xenogeneic components in that dissociation media did not contain bovine trypsin. We report equivalent or superior proliferation, and differentiation activity of WJ-MSC cultured in XcytePLUS™ media as compared to FCS.
Cells and tissue culture
Wharton’s Jelly mesenchymal stem cells were isolated from healthy donors according to Secco et al. . Briefly, umbilical cords of minimum 25 cm long were dissected, and cut in pieces of 8 cm, and they were digested using Collagenase 1.67% (Sigma C9891) during 60 min at 37°C. Tissue was washed twice and all supernatant was centrifuged to collect the pellet and then plated at 1 × 104 cells/cm2. Cells were cultured using MEM alpha (Life 32561102) supplemented with 2 mM GlutaMax (Life 35050-079) and 10% FCS (Life 16000044).
All Isolated MSCs were frozen in Passage 2. Immunophenotype characterization was performed and based on those results, four lots were selected from a stock of cells to continue the experiments.
MSCs were thawed and plated into 6 well plates (6 wells per treatment), 5 × 104 cells/well, using XcytePLUS™ (iBiologics XPGS-001-500) (5, 7.5, and 10%) or 10% FCS supplemented media in two different laboratories. Cells were grown up to 80–90% confluence, then passaged using Tryple Express (Life Technologies), splits were 1:6, up to passage 6. Cell counts were performed every passage before re-plating to determine cell-doubling time. Finally, cells were frozen for further analysis of membrane markers expression and differentiation.
Flow cytometry was performed using Guava EasyCyte Mini (Millipore) flow cytometer. Antibodies CD105, CD73, CD90, CD34, CD45 were purchased from (BD pharmingen), isotype controls for FITC and RPE were utilized.
For the statistical analysis, comparison of averages were made using GraphPad (Prism 6) software. Two-way ANOVA with Tukey test was used for doublings per day, cumulative number of cells and characterization analysis. One-way ANOVA was used for inter-laboratory analysis. In all tests a confidence interval of 95% was used.
Two lots were used for the differentiation assay. Briefly, 24 well plates were used and differentiation medium was prepared using DMEM low glucose, without phenol red (Life Technologies) supplemented with 2 mM glutaMax and 10% FCS. Adipogenesis media was supplemented with 1 μM dexamethasone, 500 μM 3-isobutyl-1-methylxantine, 60 μM indometacine, 5 μg/ml insulin. Chondrogenesis media with 0.1 μM dexamethasone, 50 μM ascorbate-2-phosphate, 1 mM sodium pyruvate, 1% ITS Premix, 10 ng/ml TGF-β1, and Osteogenesis media with 0.1 μM dexamethasone, 50 μM ascorbate-2-phosphate, 10 mM β-glycerophosphate. After 21 days, alcian blue, alizarin red and oil red O were used to stain chondrocytes, osteocytes and adipocytes, respectively.
Doubling rate superior with WJ-MSC cultured in XCyte Media versus FCS
Maintenance of MSC phenotype
The current findings provide for the first time data on reproducibility of a commercially available xenogenic free media supplement and cell dissociation technique assessed across WJ-MSC donors. Equivalent or superior induction of cell proliferation was noted, as well as retention of classical MSC surface markers for 6 passages. Reproducibility of results between two independent laboratories was demonstrated.
Currently there is substantial controversy in the area of platelet lysate based cultures with various investigators providing contradictory results. Furthermore the field is complicated by lack of widespread access to various proprietary reagents utilized in the manufacture of the platelet lysate. Specifically, conditions such as concentration of heparin, fibronectin or donor characteristics all contribute to variability of product. XcytePLUS™ media is commercially available and is based on stringent characterized donors with large lots, thereby decreasing lot to lot variability.
Even though it has been reported that platelet lysate substitution for FCS results in a lower ability to differentiate into both adipogenic and osteogenic lineages, our data does not support this finding . Due to enhanced proliferation rates with platelet lysate media, population doublings could be higher compared to FCS supplemented media. We have demonstrated that population doublings should be controlled so that the cells can retain differentiation ability.
The large-scale production of clinical grade MSCs demands a xeno-free standardized culture system. Feasibility of the use of XcytePLUS™ was demonstrated with consistent results in proliferation rate, characterization and differentiation potential. XcytePLUS™ is a pooled human platelet lysate product, which allows for a commercial therapeutic MSC product.
NHR and MM conceived and designed the experiment. NHR, MM, JR, KC, NJ, SR, NS, TEI, FS, ANP were involved in experimental design, execution of experiments, and writing of the manuscript. All authors read and approved the final manuscript.
This work was funded by internal grants from the Department of Cardiothoracic Surgery at the University of Utah, Salt Lake City, Utah.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Carrel A (1912) On the permanent life of tissues outside of the organism. J Exp Med 15(5):516–528PubMed CentralPubMedView ArticleGoogle Scholar
- Shodell M, Rubin H, Gerhart J (1972) Neutralization of growth-inhibitory material present in calf serum by conditioning factors elaborated by chick embryo cells in culture. Exp Cell Res 74(2):375–382PubMedView ArticleGoogle Scholar
- Thomas ED (1983) Bone marrow transplantation. A lifesaving applied art. An interview with E. Donnall Thomas, MD. JAMA 249(18):2528–2536PubMedView ArticleGoogle Scholar
- Selvaggi TA, Walker RE, Fleisher TA (1997) Development of antibodies to fetal calf serum with arthus-like reactions in human immunodeficiency virus-infected patients given syngeneic lymphocyte infusions. Blood 89(3):776–779PubMedGoogle Scholar
- Irie RF, Irie K, Morton DL (1974) Natural antibody in human serum to a neoantigen in human cultured cells grown in fetal bovine serum. J Natl Cancer Inst 52(4):1051–1058PubMedGoogle Scholar
- Macy E et al (1989) Anaphylaxis to infusion of autologous bone marrow: an apparent reaction to self, mediated by IgE antibody to bovine serum albumin. J Allergy Clin Immunol 83(5):871–875PubMedView ArticleGoogle Scholar
- Mackensen A, Drager R, Schlesier M, Mertelsmann R, Lindemann A (2000) Presence of IgE antibodies to bovine serum albumin in a patient developing anaphylaxis after vaccination with human peptide-pulsed dendritic cells. Cancer Immunol 49(3):152–156Google Scholar
- Kadri N, Potiron N, Ouary M, Jegou D, Gouin E, Bach JM et al (2007) Fetal calf serum-primed dendritic cells induce a strong anti-fetal calf serum immune response and diabetes protection in the non-obese diabetic mouse. Immunol Lett 108(2):129–136Google Scholar
- Forni G, Green I (1976) Heterologous sera: a target for in vitro cell-mediated cytotoxicity. J Immunol 116(6):1561–1565PubMedGoogle Scholar
- Lauer SJ, Finlan J, Borella LD, Piaskowski VD, Casper JT (1983) In vitro enhancement of peripheral blood mononuclear cell natural killer activity following short term incubation with fetal calf serum. J Clin Lab Immunol 12(2):105–110Google Scholar
- Arien-Zakay H, Lazarovici P, Nagler A (2010) Tissue regeneration potential in human umbilical cord blood. Best Pract Res Clin Haematol 23(2):291–303PubMedView ArticleGoogle Scholar
- Obermajer N, Popp FC, Johnson CL, Benseler V, Dahlke MH (2014) Rationale and prospects of mesenchymal stem cell therapy for liver transplantation. Curr Opinion Organ Transplant 19(1):60–64Google Scholar
- Hu J, Yu X, Wang Z, Wang F, Wang L, Gao H et al (2013) Long term effects of the implantation of Wharton's jelly-derived mesenchymal stem cells from the umbilical cord for newly-onset type 1 diabetes mellitus. Endocrine J 60(3):347–357Google Scholar
- Huo W, Liu X, Tan C, Han Y, Kang C, Quan W et al (2014) Stem cell transplantation for treating stroke: status, trends and development. Neural Regen Res 9(17):1643–1648. doi:10.4103/1673-5374.141793
- Kaplan JM, Youd ME, Lodie TA (2011) Immunomodulatory activity of mesenchymal stem cells. Curr Stem Cell Res Ther 6(4):297–316PubMedView ArticleGoogle Scholar
- Sundin M, Ringden O, Sundberg B, Nava S, Gotherstrom C, Le Blanc K (2007) No alloantibodies against mesenchymal stromal cells, but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell recipients. Haematologica 92(9):1208–1215Google Scholar
- Bowen-Pope DF, Ross R (1984) Platelet-derived growth factor. Clin Endocrinol Metab 13(1):191–205PubMedView ArticleGoogle Scholar
- Eastment CT, Sirbasku DA (1980) Human platelet lysate contains growth factor activities for established cell lines derived from various tissues of several species. In Vitro 16(8):694–705PubMedView ArticleGoogle Scholar
- Muller I, Kordowich S, Holzwarth C, Spano C, Isensee G, Staiber A et al (2006) Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM. Cytotherapy 8(5):437–444. doi:10.1080/14653240600920782
- Lange C, Cakiroglu F, Spiess AN, Cappallo-Obermann H, Dierlamm J, Zander AR (2007) Accelerated and safe expansion of human mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine. J Cellular Physiol 213(1):18–26. doi:10.1002/jcp.21081
- Schallmoser K, Bartmann C, Rohde E, Reinisch A, Kashofer K, Stadelmeyer E et al (2007) Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells. Transfusion 47(8):1436–1446. doi:10.1111/j.1537-2995.2007.01220.x
- Capelli C, Domenghini M, Borleri G, Bellavita P, Poma R, Carobbio A et al (2007) Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts. Bone Marrow Transpl 40(8):785–791. doi:10.1038/sj.bmt.1705798
- Carrancio S, Lopez-Holgado N, Sanchez-Guijo FM, Villaron E, Barbado V, Tabera S et al (2008) Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification. Experimental Hematol 36(8):1014–1021Google Scholar
- Salvade A, Della Mina P, Gaddi D, Gatto F, Villa A, Bigoni M et al (2010) Characterization of platelet lysate cultured mesenchymal stromal cells and their potential use in tissue-engineered osteogenic devices for the treatment of bone defects. Tissue Eng Part C 16(2):201–214. doi:10.1089/ten.TEC.2008.0572
- Bieback K, Hecker A, Kocaomer A, Lannert H, Schallmoser K, Strunk D et al (2009) Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow. Stem Cells 27(9):2331–2341Google Scholar
- Crespo-Diaz R, Behfar A, Butler GW, Padley DJ, Sarr MG, Bartunek J et al (2011) Platelet lysate consisting of a natural repair proteome supports human mesenchymal stem cell proliferation and chromosomal stability. Cell Transplant 20(6):797–811Google Scholar
- Xia W, Li H, Wang Z, Xu R, Fu Y, Zhang X et al (2011) Human platelet lysate supports ex vivo expansion and enhances osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. Cell Biol Int 35(6):639–643Google Scholar
- Kinzebach S, Bieback K (2013) Expansion of mesenchymal stem/stromal cells under xenogenic-free culture conditions. Adv Biochem Eng Biotechnol 129:33–57PubMedGoogle Scholar
- Warnke PH, Humpe A, Strunk D, Stephens S, Warnke F, Wiltfang J et al (2013) A clinically-feasible protocol for using human platelet lysate and mesenchymal stem cells in regenerative therapies. J Craniomaxillofac Surg 41(2):153–161Google Scholar
- Fekete N, Rojewski MT, Furst D, Kreja L, Ignatius A, Dausend J et al (2012) GMP-compliant isolation and large-scale expansion of bone marrow-derived MSC. PloS One 7(8):e43255Google Scholar
- Bernardi M, Albiero E, Alghisi A, Chieregato K, Lievore C, Madeo D et al (2013) Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow-derived mesenchymal stromal cells. Cytotherapy 15(8):920–929Google Scholar
- Mojica-Henshaw MP, Jacobson P, Morris J, Kelley L, Pierce J, Boyer M et al (2013) Serum-converted platelet lysate can substitute for fetal bovine serum in human mesenchymal stromal cell cultures. Cytotherapy 15(12):1458–1468Google Scholar
- Shanskii YD, Sergeeva NS, Sviridova IK, Kirakozov MS, Kirsanova VA, Akhmedova SA et al (2013) Human platelet lysate as a promising growth-stimulating additive for culturing of stem cells and other cell types. Bull Exp Biol Med 156(1):146–151Google Scholar
- Iudicone P, Fioravanti D, Bonanno G, Miceli M, Lavorino C, Totta P et al (2014) Pathogen-free, plasma-poor platelet lysate and expansion of human mesenchymal stem cells. J Transl Med 12:28Google Scholar
- Shih DT, Burnouf T (2014) Preparation, quality criteria, and properties of human blood platelet lysate supplements for ex vivo stem cell expansion. N BiotechnolGoogle Scholar
- Centeno CJ, Schultz JR, Cheever M, Robinson B, Freeman M, Marasco W (2010) Safety and complications reporting on the re-implantation of culture-expanded mesenchymal stem cells using autologous platelet lysate technique. Curr Stem Cell Res Ther 5(1):81–93Google Scholar
- Fekete N, Gadelorge M, Furst D, Maurer C, Dausend J, Fleury-Cappellesso S et al (2012) 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. Cytotherapy 14(5):540–554Google Scholar
- Abdelrazik H, Spaggiari GM, Chiossone L, Moretta L (2011) Mesenchymal stem cells expanded in human platelet lysate display a decreased inhibitory capacity on T- and NK-cell proliferation and function. Eur J Immunol 41(11):3281–3290Google Scholar
- Copland IB, Garcia MA, Waller EK, Roback JD, Galipeau J (2013) The effect of platelet lysate fibrinogen on the functionality of MSCs in immunotherapy. Biomaterials 34(32):7840–7850Google Scholar
- Horn P, Bokermann G, Cholewa D, Bork S, Walenda T, Koch C et al (2010) Impact of individual platelet lysates on isolation and growth of human mesenchymal stromal cells. Cytotherapy 12(7):888–898Google Scholar
- Lohmann M, Walenda G, Hemeda H, Joussen S, Drescher W, Jockenhoevel S et al (2012) Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells. PloS One 7(5):e37839Google Scholar
- Hemeda H, Kalz J, Walenda G, Lohmann M, Wagner W (2013) Heparin concentration is critical for cell culture with human platelet lysate. Cytotherapy 15(9):1174-1181Google Scholar
- Secco M, Zucconi E, Vieira NM, Fogaca LL, Cerqueira A, Carvalho MD et al (2008) Multipotent stem cells from umbilical cord: cord is richer than blood! Stem Cells 26(1):146–150Google Scholar
- Ben Azouna N, Jenhani F, Regaya Z, Berraeis L, Ben Othman T, Ducrocq E et al (2012) Phenotypical and functional characteristics of mesenchymal stem cells from bone marrow: comparison of culture using different media supplemented with human platelet lysate or fetal bovine serum. Stem Cell Res Ther 3(1):6Google Scholar