Cytotechnology (2021) 73:513–522 https://doi.org/10.1007/s10616-021-00474-3 (0123456789().,-volV) ( 01234567 89().,-volV) METHODS PAPER Towards the standardization of methods of tissue processing for the isolation of mesenchymal stromal cells for clinical use Elisabeth Garcı́a-Muñoz . Joaquim Vives Received: 14 January 2021 / Accepted: 4 May 2021 / Published online: 10 May 2021 Ó The Author(s) 2021 Abstract Multipotent mesenchymal stromal cells such as rejection by the host. This shift to the (MSCs) are currently the most extensively studied allogeneic setting entails the need for high production type of adult stem cells in advanced stages of of MSCs to ensure availability of sufficient cell development in the field of regenerative medicine. numbers for transplantation, and therefore making The biological properties of MSCs have generated the search for alternative tissue sources of highly great hope for their therapeutic use in degenerative and proliferative MSC cultures with low levels of senes- autoimmune conditions that, at present, lack effective cence occurrence, which is one of the greatest current treatment options. Over the last decades, MSCs have challenges in the scale up of therapeutic cell biopro- been typically obtained from adult bone marrow, but cessing. Herein we (i) present the main isolation the extraction process is highly invasive and the protocols of MSCs from bone marrow, adipose tissue quality and numbers of isolated cells is drastically and Wharton’s jelly of the umbilical cord; and (ii) influenced by patient age, medication and associated compare their qualities from a bioprocess standpoint, comorbidities. Therefore, there is currently an open addressing both quality and regulatory aspects, in view discussion on the convenience of allogeneic over of their anticipated clinical use. autologous treatments, despite potential disadvantages Keywords Multipotent mesenchymal stromal cells  Bone marrow  Wharton’s jelly  Umbilical cord  E. Garcı́a-Muñoz  J. Vives (&) Adipose tissue  MSCs isolation protocols Banc de Sang iTeixits, Edifici Dr. Frederic Duran i Jordà, Passeig Taulat, 116, 08005 Barcelona, Spain e-mail: jvives@bst.cat E. Garcı́a-Muñoz e-mail: eliigm@outlook.com Introduction J. Vives Mesenchymal stromal cells (MSCs) are multipotent Musculoskeletal Tissue Engineering Group, Vall D’Hebron Research Institute (VHIR), Universitat progenitor cells of mesodermal origin, which are able Autònoma de Barcelona, Passeig de la Vall d’Hebron to self-renew and differentiate into different special- 129-139, 08035 Barcelona, Spain ized cell lineages of skeletal tissues, such as: chon- drocytes (cartilage cells), osteoblasts (bone cells) and J. Vives Departament de Medicina, Universitat Autònoma de adipocytes (fat cells) (Nombela-Arrieta et al. 2011). Barcelona, Passeig de la Vall d’Hebron 129-139, They were first isolated from the bone marrow, 08035 Barcelona, Spain 123 514 Cytotechnology (2021) 73:513–522 although they have been also found in a wide variety of number of progenitor cells (Priya et al. 2014). adult and neonatal tissues such as adipose tissue, Historically, bone marrow (BM) has been the pre- lungs, dental pulp, peripheral blood, placenta and ferred source of MSCs for clinical use, and this is umbilical cord (both from blood and tissue), among indeed the best-studied source in the last decades others (Sanz et al. 2016; Amable et al. 2014; Kern et al. (Friedenstein et al. 1968). However, BM harvesting is 2006; Wagner et al. 2005). In fact, recent studies a highly invasive procedure as it requires anaesthesia, evidenced that MSCs lie adjacent to blood vessels and can cause post-operative pain or discomfort, and most likely derive from vascular mural cells known as exposes the patient to a high risk of nosocomial pericytes, therefore suggesting that MSC could result infections (Naji et al. 2019). Furthermore, it is from any vascularized tissue source (Nombela-Arrieta estimated that MSCs represent only 0.001–0.01% of et al. 2011). The identity of MSC cultures is generally all nucleated cells present in the bone marrow (Sanz verified by compliance with a set of characteristics et al. 2016), and their proliferation capacity decline known as the ‘‘minimal criteria’’ established by the with age of the donor. More recently, adipose tissue International Society for Cell & Gene Therapy, (AT) and the Wharton’s jelly (WJ) of the umbilical including: (i) plastic-adherence of cells that are cord have become alternative sources of MSCs, since maintained under standard culture conditions; (ii) they are associated to minimally or even non-invasive expression (C 95%) of CD105, CD73 and CD90 procedures (Amable et al. 2014; Kern et al. 2006; Naji markers and lack (B 2%) of CD45, CD34, CD14 or et al. 2019). The extraction of MSCs from adipose CD11b, CD79a o CD19 and HLA-DR markers; (iii) tissue is performed by liposuction, the most practiced in vitro differentiation potential into osteoblastic, cosmetic procedure nowadays. Despite requiring chondrogenic and adipoblastic lineages (Dominici anaesthesia, this is considered a safe and painless et al. 2006). MSCs have made their mark in the field of process with a minimal risk of complications for the cell therapy as promising candidates for the treatment patient (Haeck et al. 2009). In addition, a greater of a wide array of diseases, specifically those of quantity of starting sample can be obtained, and 1–5% degenerative and immunological origin (Naji et al. of cells correspond to MSC populations (Naji et al. 2019). MSCs possess five fundamental biological 2019). On the other hand, the frequency of MSC properties that make them attractive for clinical populations in the umbilical cord is similar or lower application: (i) migration into inflamed sites of than the frequency found in the bone marrow but, in damaged/diseased tissues when injected intra- return, they present enhanced expansion potential venously, (ii) promotion of a local immunosuppres- in vitro due to its foetal origin (Naji et al. 2019). sive environment, (iii) trophic function through the Moreover, its sourcing does not represent any harm to secretion of bioactive molecules that stimulate tissue the mother, the foetus or the patient. Medical history of healing, (iv) self-renewal and expansion capacity, and donors has an impact on the qualities of MSC and (v) differentiation into various specialised cell types (Fig. 1). Importantly, clinical use of MSC has been reported to be safe irrespective of the tissue source, route of administration, dose and condition treated (Thompson et al. 2020; Lalu et al. 2012, 2018). Detailed analyses of the identity, secretome and potency of MSC from different tissues have been widely described in the literature, including back to back comparisons both in vitro and in vivo (Hass et al. 2011; Grégoire et al. 2019). However, even minor changes in the bioprocess design or media formulation can impact on the attributes of MSCs and ultimately on their activity in the patient (Wagner et al. 2007). Fig. 1 Biological properties of MSC of clinical interest include: The ideal source of MSCs for clinical use must be (1) trophic function, (2) homing function, (3) immunomodula- abundant, easily accessible in order to minimise tory function, (4) multipotency function and (5) self-renewal invasiveness and morbidity, and contain a high and proliferation function 123 Cytotechnology (2021) 73:513–522 515 therefore neonatal sources such as the umbilical cord assessment includes the study of the persistence of could result in healthier cells for allogeneic use. cells, ectopic differentiation and biodistribution, in Herein we present and discuss the existing infor- compliance with the principles of good laboratory mation on different isolation protocols of MSC practice (GLP) (Vives et al. 2019). Upon approval by progenitors derived from BM, AT and WJ. the competent authority (i.e. Agencia Española de Medicamentos y Productos Santiarios, AEMPS, in Spain), clinical-grade production of MSC is per- Regulatory requirements for tissue sourcing formed in clean rooms following procedures compli- ant with current good manufacturing practice (GMP), As it happens to any other advanced therapy medicinal and human clinical trials are conducted under good product, the development of MSC-based therapies clinical practice (GCP) after approval by the Ethics requires strict adherence to regulatory and quality Committee and conducted in accordance with the standards (Schneider et al. 2010; Vives et al. 2015). Declaration of Helsinki (Gastelurrutia et al. 2021). In The key steps of such process are summarised phase IV, marketing authorization of the MSC-based schematically in Fig. 2. First, cell and tissue donation product is granted by competent authorities (cen- must be approved by the local ethics committee and tralised by the European Medicines Agency, EMA, in the competent authority (i.e. Organització Catalana de Europe). Alternatively, products under investigation Transplantaments, OCATT, in our territory). Next, in can be authorized case-by-case for compassionate uses the preclinical R & D phase, a first evaluation of the or, those that are not industrially prepared and are not efficacy, safety and feasibility of the treatment is intended for marketing could be used under the performed through a series of proof-of-concept studies hospital exemption clause for the treatment of specific either in vitro, in vivo or both. Further safety conditions in approved centres (Roura et al. 2017; Fig. 2 Development programme for cell-based medicinal products. GLP good laboratory practices, GMP good manufacturing practices, GCP good clinical practices, GDP good distribution practices 123 516 Cytotechnology (2021) 73:513–522 Coppens et al. 2020; Cuende et al. 2014; Hills et al. Through this technique, initial cultures present a high 2020). hematopoietic contamination (i.e. monocytes), but the MSC fraction is efficiently enriched over subsequent passaging (Bieback et al. 2008). This methodology MSC isolation techniques was successfully used in our laboratory for the production of BM-MSC in a Phase I/IIa clinical trial Isolation of bone marrow-derived MSCs (BM- of spinal fusion in degenerative disk disease (Garcı́a MSCs) de Frutos et al. 2020; Vives et al. 2020). BM-MSCs for clinical use are derived from progenitor Isolation of adipose tissue-derived MSCs (ADSC) cells typically isolated from total bone marrow aspirates obtained by puncturing the iliac crest of ADSC are isolated under anaesthesia by liposuction or donors under local anaesthesia. Otherwise total bone lipectomy of the subcutaneous fat tissue located in marrow can also be obtained from osteotomy of the areas such as the abdomen or the gluteus (Priya et al. femoral shafts during orthopaedic surgical procedures 2014; Ghorbani et al. 2014). (Kern et al. 2006; Wagner et al. 2005; Naji et al. 2019; Enzymatic digestion: the enzymatic digestion is the Hua et al. 2014). The fraction of progenitor cells is conventional technique used for the isolation of ADSC further enriched by following either one of the two for clinical application. This method involves exten- methods detailed next: sive washes of the lipoaspirate to remove other blood Density gradient centrifugation: a wide variety of cells, cellular debris and oil, and an enzymatic protocols describing the isolation of total bone mar- treatment of the resulting adipose fraction at 37 °C row-derived MSCs involve enriching techniques, such under permanent or intermittent shaking (Amable as density gradient centrifugation of BM aspirates and et al. 2014; Kern et al. 2006; Wagner et al. 2005; Wang later collection of the mononuclear cell fraction, in et al. 2016). Collagenase is the most widely used which MSCs are present together with other types of enzyme, but its concentration and incubation time can progenitor cells (Kern et al. 2006; Wagner et al. 2005; vary according to the protocol (Nombela-Arrieta et al. Hua et al. 2014; Bieback et al. 2008; Jin et al. 2013). 2011; Amable et al. 2014; Wagner et al. 2005; Zuk Most methods currently in use employ density gradi- et al. 2001). Once the digestion process is fully ent media such as Percoll and Ficoll (Bieback et al. completed and the enzyme neutralized, further cen- 2008). Percoll are silica-based media with a density of trifugation steps are performed to pellet down the 1.130 g/mL that can be adjusted to obtain solutions of stromal vascular fraction (SVF) that contains a different density. Ficoll is a registered trademark heterogeneous mixture of cells including mesenchy- solution with a density of 1.077 g/mL ready-to-use for mal progenitors. Adherent ADSC are separated from the isolation of mononuclear cells from human all the remaining non-adherent cells by extensive peripheral blood (Munteanu et al. 2005). Interestingly, washings after the initial adherence step. As an these methods can be automated with the Sepax device illustrative example, collagenase treatment was used (Biosafe, Cytiva) using Ficoll-Paque reagent (Cytiva). in the production of ADSC for the treatment of To illustrate this application, we followed this complex cryptoglandular perianal fistula by Garcia- methodology in the first step for the production of Arranz and collaborators (Garcia-Arranz et al. 2020). BM-MSC used in a Phase I/IIa clinical trial for the Explant culture: recent studies introduce explant treatment of gonarthrosis (Codinach et al. 2016; Soler culture as an enzyme-free method for the isolation of et al. 2016). ADSC (Priya et al. 2014; Ghorbani et al. 2014). Direct plating: although several new devices are Briefly, the lipoaspirate is extensively washed and available commercial to enrich the mesenchymal cell allowed to settle down for 5 min to separate the fraction (Table 2), a very simple protocol for BM- adipose fraction from the aqueous phase. Fat is MSC isolation is based on their ability to adhere to the mechanically fractionated by cutting it into small culture plates, by following a direct plating strategy of pieces that are seeded in a culture plate with basal total bone marrow aspirates to separate plastic adher- medium at 37 °C with 5% humidified CO2. Explants ent MSCs from non-adherent hematopoietic cells. are removed once cells have migrated from the explant 123 Cytotechnology (2021) 73:513–522 517 tissue to the culture plate. As far as we know, this seeded in a culture plate with basal medium (Wang method is only described in research studies and has et al. 2016; Salehinejad et al. 2012). Explants are not yet been used in any clinical protocol. maintained in a humidified atmosphere with 5% CO2 at 37 °C, until cells migrate from the tissue to the culture plate. After that, explants are removed and Isolation of Wharton’s jelly-derived MSCs (WJ- cultures preserved until they reach 90% confluence MSCs) (Hua et al. 2014; Salehinejad et al. 2012; Han et al. 2013; Ishige et al. 2009). Specifically, we have WJ-MSCs are obtained from the donation of umbilical investigated the safety and efficacy of WJ-MSC cords of healthy full-term neonates, after receiving the resulting from the use of this methodology in clinical informed consent of the parents (Salehinejad et al. trials for the treatment of inflammatory conditions 2012; Han et al. 2013). Similar to bone marrow and including chronic spinal cord injury (ClinicalTrials.- fat, different methods have been developed for the gov Id. NCT03003364), severe respiratory distress isolation of mesenchymal progenitors. Remarkably, in due to SARS-CoV-2 infection (ClinicalTrials.gov Id. all cases, ex vivo expanded MSC appear to be NCT04390139) and myocardial infarction in the equivalent in terms of viability, morphology, rate of PERISCOPE (the PERIcardial matrix with mesenchy- proliferation, surface marker expression, levels of mal Stem Cells fOr the treatment of PatiEnts with cytokine secretion and differentiation capacity (Skiles infarcted myocardial tissue) trial (ClinicalTrials.gov et al. 2020). Id. NCT03798353) (Albu et al. 2021; Prat-Vidal et al. Enzymatic digestion: this technique involves the 2020). enzymatic digestion at 37 °C, 5% CO2 in a humidified incubator, of umbilical cord fragments (3–5 cm) previously washed after arteries and blood vessel are Attributes of adult and neonatal MSCs removed (Amable et al. 2014; Hua et al. 2014; Wang et al. 2016; Salehinejad et al. 2012; Han et al. 2013). Several studies have shown that MSCs derived from Although collagenase is the most widely used enzyme, bone marrow, adipose tissue and Wharton’s jelly share many laboratories also use trypsin, hyaluronidase or a a very similar immunoprofile that complies with the combination (Hua et al. 2014; Salehinejad et al. 2012). ISCT’s minimal criteria: they are positive for CD105, The digestion process conditions may vary according CD73 and CD90 and do not present CD45, CD34, to the protocol, mostly subjected to the enzyme type CD14 or CD11b, CD79a o CD19 and HLA-DR and concentration employed. Anyhow, once the markers (Amable et al. 2014; Kern et al. 2006; Hua enzymatic digestion is finished, WJ-MSCs are isolated et al. 2014; Jin et al. 2013; Wang et al. 2016; Lee et al. by adherence, after culturing the resulting pellet. To 2004; Heo et al. 2016). All of them also possess our knowledge, this technique has only been described multilineage differentiation capacity (Amable et al. in research studies and has not yet been reported in any 2014; Kern et al. 2006; Wagner et al. 2005; Jin et al. clinical protocol. However, the MERLIN (MEsenchy- 2013; Wang et al. 2016), although WJ-MSCs appear to mal stem cells to Reduce Liver INflammationlarger take longer times to differentiate in vitro towards fragments of the umbilical cord tissue (including WJ) osteogenic and adipogenic lineages compared to adult trial reported the use of the enzymatic methods for the BM-MSCs and ADSC (Amable et al. 2014). However, preparation of the Master Cell Bank of MSC from we observed that factors secreted by BM-MSCs during larger fragments of umbilical tissue, therefore poten- osteogenic differentiation greatly accelerate osteo- tially including the Wharton’s Jelly (ClinicalTrials.- genic induction of WJ-MSCs, suggesting that the gov Id. NCT02997878). intra-osseous environment could be sufficient for this Explant culture: through this method, WJ-MSCs population to guarantee a successful bone regenera- are isolated without the need of any enzymatic tion, with a similar therapeutic efficacy to that of BM- treatment. In short, umbilical cord fragments previ- MSCs (Cabrera-Pérez et al. 2019). Although WJ- ously washed and with the arteries and blood vessel MSCs may initially take longer times to reach 80–90% removed, are mechanically fractionated by cutting confluence (Hua et al. 2014; Salehinejad et al. 2012; them into smaller fragments (2–3 mm) that are directly Han et al. 2013; Nagamura-Inoue et al. 2014; Cheng 123 518 Cytotechnology (2021) 73:513–522 et al. 2011), they are typically cultured for more Due to the intense degradation of the tissue, cultures passages before presenting signs of senescence from enzymatic digestions are very heterogeneous at (Table 1), with higher population doubling levels in initial passages with increased hematopoietic contam- comparison to adult BM-MSCs and ADSC (Amable ination (as a higher percentage of CD34 and HLA-DR et al. 2014; Hua et al. 2014; Cheng et al. 2011). Indeed, positive cells are detected) (Priya et al. 2014; tissue age seems to be a key factor in the proliferative Salehinejad et al. 2012), are morphologically affected and expansion capacity of the isolated MSCs, being and have lower proliferation rates (Hua et al. 2014; greater the more primitive the tissue is (Stolzing et al. Salehinejad et al. 2012). Therefore, from a clinical and 2008; Jones et al. 2010). This fact was further regulatory point of view, explant culture would corroborated by Wang and collaborators (2016), who present several advantages comparison to enzymatic showed that MSCs derived from foetal bone marrow, a digestions (Table 2). However, more studies are tissue even more primitive than the umbilical cord, needed involving different enzyme types, concentra- present higher rates of proliferation and expansion tions and incubation times, since enzymatic digestions than WJ-MSCs. not affecting cell adhesion and viability could lead to highly efficient cultures (Priya et al. 2014; Han et al. 2013), which is a key point for large-scale MSCs Comparison of isolation methods: explant culture isolation and expansion. Having said this, the addition vs. enzymatic digestion of enzymes (mostly from xenogeneic origin) may pose safety risks that must to be assessed thoroughly before There is still no consensus that determines the most submission of the Investigational Medicinal product cost-effective protocol for the isolation of MSCs Dossier (IMPD) to the regulatory authority (Vives populations. Explant culture prolongs primary culture et al. 2021). time, but it is an inexpensive process requiring very little manipulation and resulting in homogeneous cell populations, since only those that can migrate from the Protocol standardization and optimization needs tissue to the plastic will proliferate (Priya et al. 2014; Hua et al. 2014; Ghorbani et al. 2014; Salehinejad Leaving aside differences in MSCs properties accord- et al. 2012; Han et al. 2013). On the other hand, ing to the tissue source, there are several other factors enzymatic digestion allows rapid cell isolation, but that may impact on the variability of their ultimate enzymes increase the price of the process and generate functional characteristics such as the isolation method a very high proteolytic stress that may affect cell and MSC enrichment techniques, formulations of the membranes and consequently, their adhesion capacity expansion culture medium (considering the use of and viability (Hua et al. 2014; Salehinejad et al. 2012). animal derived supplements or xeno-free/serum-free Moreover, this technique requires multiple steps chemically defined media), environmental conditions leading to a higher risk of biological contamination. and scale up strategy (Garcı́a-Fernández et al. 2020; Table 1 BM-MSCs, ADSC and WJ-MSCs isolation and expansion characteristics MSC source Average time to 80–90% confluency No. passages Max No. passage BM 7 days P8–P13 (adult) P22–P24 (foetal) P13 (adult) AT 10–14 days (Exp) P8–P13 P20 3–7 days (Enz) WJ 2–4 weeks (Exp) P17–P18 P25 12–14 days (Enz) WJ-MSCs and foetal BM-MSCs present the highest proliferation capacity. Enzymatic digestion allows faster ADSC and WJ-MSCs isolation compared to the explant culture technique BM bone marrow, AT adipose tissue, WJ Wharton’s jelly, Exp xplant culture method, Enz enzymatic digestion method, P passage 123 Cytotechnology (2021) 73:513–522 519 Table 2 Comparison of MSC isolation methods, which can benefit from MSC enrichment techniques MSC isolation Advantages Disadvantages technique Enzymatic Rapid MSC isolation Increased price digestion High proteolytic stress on cells Increased hematopoietic contamination Several processing steps: high risk of biological contaminations Explant culture High proliferation rate Time-consuming Cheaper process Do not cause cell membranes damage Homogenous populations Little manipulation: low risk of biological contaminations Enrichment Enrichment of the MSC fraction Additional step at the start of the scale up production workflow techniques Fast process Additional cell manipulation: increased risk of contaminations and addition of impurities Automated (in some cases) Expensive Galipeau 2013). For this reason, specifications of final cumulative population doublings were always under product need to be clearly established in terms of 40, which is considered the safety limit in culture. cellular identity, purity and potency according to the expected mechanism of action (Vives et al. 2019). There is currently a lack of global harmonisation in Conclusions compliance with quality standards and regulatory guidelines for the establishment of GMP-compliant In summary, all three types of MSC cultures deriving manufacturing protocols to ensure scalable, robust and from bone marrow, adipose tissue and Wharton’s jelly reproducible production of MSCs that satisfies safety, meet the minimum requirements established by the quality and quantity demands for its therapeutic ISCT, that is: they are spindle-shaped adherent cells, application therefore ensuring cross-comparison of they accomplish the established immunogenic profile, data resulting from clinical trials using MSC-based and they have the ability to differentiate into products prepared in different laboratories (Naji et al. osteoblasts, adipocytes, and chondrocytes. However, 2019; Bieback et al. 2008; Garcı́a-Fernández et al. choosing the best source of MSCs by directly 2020). Our contribution to ensure consistency of the comparing the three population characteristics qualities of MSC in clinical trials is the development remains a very difficult task, since a standardized and validation of a robust bioprocess design for the isolation protocol has not yet been defined, and the scale up of clinical grade WJ-MSC by following a initial source may not be the only factor that influences two-tiered cell banking strategy (Master Cell Bank the basic properties of resulting MSCs. Consequently, and Working Cell Bank) in compliance with GMP additional comparative studies involving different (Grau-Vorster et al. 2019; Oliver-Vila et al. 2016). isolation conditions are needed to promote the devel- This methodology has resulted in MSC-based prod- opment of global standards and manufacturing guide- ucts in conformity with the same specifications that lines. Despite of this, the Wharton’s jelly from the were used in clinical trials and compassionate cases umbilical cord hold high expectations and appears to (Albu et al 2021; Prat-Vidal et al. 2020). Moreover, be close to an ideal source for the development of risks such as cell aging, and therefore preservation of allogeneic cell therapies, as its sourcing does not pose MSC’s attributes, were mitigated by ensuring that any risk to the mother nor the neonate, and gives rise to more primitive and hypoimmunogenic MSC 123 520 Cytotechnology (2021) 73:513–522 populations with high proliferation capacity and low Amable PR, Teixeira MVT, Carias RBV, Granjeiro JM, Boro- senescence rates. Nevertheless, Wharton’s jelly jevic R (2014) Protein synthesis and secretion in human mesenchymal cells derived from bone marrow, adipose remains the most time-consuming source of primary tissue and Wharton’s jelly. Stem Cell Res Ther 5:1–13. culture MSCs isolation. The establishment of cost- https://doi.org/10.1186/scrt442 efficient protocols that allow large-scale expansion of Bieback K, Schallmoser K, Klüter H, Strunk D (2008) Clinical WJ-MSCs without altering their biological properties, protocols for the isolation and expansion of mesenchymal stromal cells. Transfus Med Hemother 35:286–294. https:// is one of the greatest challenges to ensure the doi.org/10.1159/000141567 progression of this cell type towards cell therapies. Cabrera-Pérez R, Monguió-Tortajada M, Gámez-Valero A et al (2019) Osteogenic commitment of Wharton’s jelly mes- Acknowledgements We are grateful to the Generalitat de enchymal stromal cells: mechanisms and implications for Catalunya that awarded our laboratory as Consolidated bioprocess development and clinical application. Stem Cell Research Group (Ref. 2017SGR719). Res Ther 10:1–11. https://doi.org/10.1186/s13287-019- 1450-3 Author Contributions EGM and JV Conceptualization; EGM Cheng H, Qiu L, Ma J et al (2011) Replicative senescence of and JV methodology; EG and JV formal analysis; EGM and JV human bone marrow and umbilical cord derived mes- investigation; EGM and JV writing—original draft preparation; enchymal stem cells and their differentiation to adipocytes EGM and JV writing—review and editing; EGM visualization; and osteoblasts. Mol Biol Rep 38:5161–5168. https://doi. JV supervision. All authors have read and agreed to the org/10.1007/s11033-010-0665-2 published version of the manuscript. Codinach M, Blanco M, Ortega I et al (2016) Design and vali- dation of a consistent and reproducible manufacture pro- cess for the production of clinical-grade bone marrow– Funding This work has been developed in the context of the derived multipotent mesenchymal stromal cells. Spanish Cell Therapy Network (TerCel, expedient number: Cytotherapy 18:1197–1208. https://doi.org/10.1016/j.jcyt. RD16/0011/0028) and project PI19/01788, which is funded by 2016.05.012 Instituto de Salud Carlos III and co-funded by European Union Coppens DGM, Hoekman J, De Bruin ML et al (2020) (ERDF/ESF)—A way to build Europe. Advanced therapy medicinal product manufacturing under the hospital exemption and other exemption pathways in Declarations seven European Union countries. Cytotherapy 22:592–600. https://doi.org/10.1016/j.jcyt.2020.04.092 Conflicts of interest The authors declare no conflict of Cuende N, Boniface C, Bravery C et al (2014) The puzzling interest. The funders had no role in the design of the study, in the situation of hospital exemption for advanced therapy writing of the manuscript, or in the decision to publish the medicinal products in Europe and stakeholders’ concerns. results. Cytotherapy 16:1597–1600. https://doi.org/10.1016/j.jcyt. 2014.08.007 Open Access This article is licensed under a Creative Com- Dominici M, Le Blanc K, Mueller I et al (2006) Minimal criteria mons Attribution 4.0 International License, which permits use, for defining multipotent mesenchymal stromal cells. The sharing, adaptation, distribution and reproduction in any med- international society for cellular therapy position state- ium or format, as long as you give appropriate credit to the ment. Cytotherapy 8:315–317. https://doi.org/10.1080/ original author(s) and the source, provide a link to the Creative 14653240600855905 Commons licence, and indicate if changes were made. The Friedenstein AJ, Petrakova KV, Kurolesova AI, Frolova GP images or other third party material in this article are included in (1968) Heterotopic of bone marrow. Analysis of precursor the article’s Creative Commons licence, unless indicated cells for osteogenic and hematopoietic tissues. Transplan- otherwise in a credit line to the material. If material is not tation 6:230–247 included in the article’s Creative Commons licence and your Galipeau J (2013) The mesenchymal stromal cells dilemma— intended use is not permitted by statutory regulation or exceeds does a negative phase III trial of random donor mes- the permitted use, you will need to obtain permission directly enchymal stromal cells in steroid-resistant graft-versus- from the copyright holder. To view a copy of this licence, visit host disease represent a death knell or a bump in the road? http://creativecommons.org/licenses/by/4.0/. Cytotherapy 15:2–8. https://doi.org/10.1016/j.jcyt.2012. 10.002 Garcı́a de Frutos A, González-Tartière P, Coll Bonet R et al References (2020) Randomized clinical trial: expanded autologous bone marrow mesenchymal cells combined with allogeneic bone tissue, compared with autologous iliac crest graft in Albu S, Kumru H, Coll R et al (2021) Clinical effects of lumbar fusion surgery. Spine J 20:1899–1910. https://doi. intrathecal administration of expanded Wharton jelly org/10.1016/j.spinee.2020.07.014 mesenchymal stromal cells in patients with chronic com- Garcia-Arranz M, Garcia-Olmo D, Herreros MD et al (2020) plete spinal cord injury: a randomized controlled study. Autologous adipose-derived stem cells for the treatment of Cytotherapy 23:146–156. https://doi.org/10.1016/j.jcyt. complex cryptoglandular perianal fistula: a randomized 2020.08.008 123 Cytotechnology (2021) 73:513–522 521 clinical trial with long-term follow-up. Stem Cells Transl Jin HJ, Bae YK, Kim M et al (2013) Comparative analysis of Med 9:295–301. https://doi.org/10.1002/sctm.19-0271 human mesenchymal stem cells from bone marrow, adi- Garcı́a-Fernández C, López-Fernández A, Borrós S, Lecina M, pose tissue, and umbilical cord blood as sources of cell Vives J (2020) Strategies for large-scale expansion of therapy. Int J Mol Sci 14:17986–18001. https://doi.org/10. clinical-grade human multipotent mesenchymal stromal 3390/ijms140917986 cells. Biochem Eng J. https://doi.org/10.1016/j.bej.2020. Jones E, English A, Churchman SM et al (2010) Large-scale 107601 extraction and characterization of CD271? multipotential Gastelurrutia P, Prat-Vidal C, Vives J, Coll R, Bayès-Genı́s A, stromal cells from trabecular bone in health and Gálvez-Montón C (2021) Transitioning from preclinical osteoarthritis: implications for bone regeneration strategies evidence to advanced therapy medicinal product: a Spanish based on uncultured or minimally cultured multipotential experience. Front Cardiovasc Med. https://doi.org/10. stromal cells. Arthritis Rheum 62:1944–54. https://doi.org/ 3389/fcvm.2021.604434 10.1002/art.27451 Ghorbani A, Jalali SA, Varedi M (2014) Isolation of adipose Kern S, Eichler H, Stoeve J, Klüter H, Bieback K (2006) tissue mesenchymal stem cells without tissue destruction: a Comparative analysis of mesenchymal stem cells from non-enzymatic method. Tissue Cell 46:54–58. https://doi. bone marrow, umbilical cord blood, or adipose tissue. Stem org/10.1016/j.tice.2013.11.002 Cells 24:1294–1301. https://doi.org/10.1634/stemcells. Grau-Vorster M, Rodrı́guez L, del Mazo-Barbara A et al (2019) 2005-0342 Compliance with good manufacturing practice in the Lalu MM, McIntyre L, Pugliese C et al (2012) Safety of cell assessment of immunomodulation potential of clinical therapy with mesenchymal stromal cells (SafeCell): a grade multipotent mesenchymal stromal cells derived from systematic review and meta-analysis of clinical trials. Wharton’s jelly. Cells 8:484. https://doi.org/10.3390/ PLoS ONE. https://doi.org/10.1371/journal.pone.0047559 cells8050484 Lalu MM, Mazzarello S, Zlepnig J et al (2018) Safety and Grégoire C, Ritacco C, Hannon M et al (2019) Comparison of efficacy of adult stem cell therapy for acute myocardial mesenchymal stromal cells from different origins for the infarction and ischemic heart failure (SafeCell Heart): a treatment of graft-vs.-host-disease in a humanized mouse systematic review and meta-analysis. Stem Cells Transl model. Front Immunol 10:1–16. https://doi.org/10.3389/ Med 7:857–866. https://doi.org/10.1002/sctm.18-0120 fimmu.2019.00619 Lee RH, Kim BC, Choi IS et al (2004) Characterization and Haeck PC, Swanson JA, Gutowski KA et al (2009) Evidence- expression analysis of mesenchymal stem cells from based patient safety advisory: liposuction. Plast Reconstr human bone marrow and adipose tissue. Cell Physiol Surg 124:28–44. https://doi.org/10.1097/prs. Biochem 14:311–324. https://doi.org/10.1159/000080341 0b013e3181b52fcd Munteanu LS, Dinu A (2005) Fractionation of Granulocytes Han YF, Tao R, Sun TJ, Chai JK, Xu G, Liu J (2013) Opti- From Whole Human Blood By Centrifugation. Practical mization of human umbilical cord mesenchymal stem cell Hints Rom J Biophys 14:1–4 isolation and culture methods. Cytotechnology Nagamura-Inoue T, He H (2014) Umbilical cord-derived mes- 65:819–827. https://doi.org/10.1007/s10616-012-9528-0 enchymal stem cells: their advantages and potential clinical Hass R, Kasper C, Böhm S et al (2011) Different populations utility. World J Stem Cells 6:195–202. https://doi.org/10. and sources of human mesenchymal stem cells (MSC): a 4252/wjsc.v6.i2.195 comparison of adult and neonatal tissue-derived MSC. Cell Naji A, Eitoku M, Favier B, Deschaseaux F, Rouas-Freiss N, Commun Signal 9:12. https://doi.org/10.1186/1478-811X- Suganuma N (2019) Biological functions of mesenchymal 9-12 stem cells and clinical implications. Cell Mol Life Sci Heo JS, Choi Y, Kim HS, Kim HO (2016) Comparison of 76:3323–3348. https://doi.org/10.1007/s00018-019- molecular profiles of human mesenchymal stem cells 03125-1 derived from bone marrow, umbilical cord blood, placenta Nombela-Arrieta C, Ritz J, Silberstein LE (2011) The elusive and adipose tissue. Int J Mol Med 37:115–125. https://doi. nature and function of mesenchymal stem cells. Nat Rev org/10.3892/ijmm.2015.2413 Mol Cell Biol 12:126–131. https://doi.org/10.1038/ Hills A, Awigena-Cook J, Genenz K et al (2020) An assessment nrm3049 of the hospital exemption landscape across European Oliver-Vila I, Coca MI, Grau-Vorster M et al (2016) Evaluation Member States: regulatory frameworks, use and impact. of a cell-banking strategy for the production of clinical Cytotherapy 22:772-779.e1. https://doi.org/10.1016/j.jcyt. grade mesenchymal stromal cells from Wharton’s jelly. 2020.08.011 Cytotherapy 18:25–35. https://doi.org/10.1016/j.jcyt.2015. Hua J, Gong J, Meng H et al (2014) Comparison of different 10.001 methods for the isolation of mesenchymal stem cells from Prat-Vidal C, Rodrı́guez-Gómez L, Aylagas M et al (2020) umbilical cord matrix: proliferation and multilineage dif- First-in-human PeriCord cardiac bioimplant: scalability ferentiation as compared to mesenchymal stem cells from and GMP manufacturing of an allogeneic engineered tissue umbilical cord blood and bone marrow. Cell Biol Int graft. EBioMedicine. https://doi.org/10.1016/j.ebiom. 38:198–210. https://doi.org/10.1002/cbin.10188 2020.102729 Ishige I, Nagamura-Inoue T, Honda MJ et al (2009) Comparison Priya N, Sarcar S, Majumdar AS, SundarRaj S (2014) Explant of mesenchymal stem cells derived from arterial, venous, culture: a simple, reproducible, efficient and economic and Wharton’s jelly explants of human umbilical cord. Int J technique for isolation of mesenchymal stromal cells from Hematol 90:261–269. https://doi.org/10.1007/s12185-009- human adipose tissue and lipoaspirate. J Tissue Eng Regen 0377-3 Med 8:706–716. https://doi.org/10.1002/term.1569 123 522 Cytotechnology (2021) 73:513–522 Roura S, Gálvez-Montón C, Mirabel C, Vives J, Bayes-Genis A Quality management and accreditation in hematopoietic (2017) Mesenchymal stem cells for cardiac repair: are the stem cell transplantation and cellular therapy. Springer, actors ready for the clinical scenario? Stem Cell Res Ther Cham 8:1–11. https://doi.org/10.1186/s13287-017-0695-y Vives J, Mirabel C (2019) Multipotent mesenchymal stromal Salehinejad P, Banu Alitheen N, Ali AM et al (2012) Compar- cells from bone marrow for current and potential clinical ison of different methods for the isolation of mesenchymal applications. Encycl Tissue Eng Regen Med. https://doi. stem cells from human umbilical cord Wharton’s jelly. Vitr org/10.1016/B978-0-12-801238-3.65506-X Cell Dev Biol Anim 48:75–83. https://doi.org/10.1007/ Vives J, Oliver-Vila I, Pla A (2015) Quality compliance in the s11626-011-9480-x shift from cell transplantation to cell therapy in non- Sanz C, Creane M, Bianco P (2016) Mesenchymal stem cells: pharma environments. Cytotherapy 17:1009–1014. https:// the ‘other’ bone marrow stem cells. EuroStemCell. https:// doi.org/10.1016/j.jcyt.2015.02.002 www.eurostemcell.org/mesenchymal-stem-cells-other- Vives J, Rodrı́guez L, Coca MI, Reales L, Cabrera-Pérez R, bone-marrow-stem-cells. Accessed 07 Jan 2021. Martorell L (2020) Use of multipotent mesenchymal stro- Schneider CK, Celis P, Salmikangas P et al (2010) Challenges mal cells fibrin and scaffolds in the production of clinical with advanced therapy medicinal products and how to meet grade bone tissue engineering products. In: Clifton NJ (ed) them. Nat Rev Drug Discov 9:195–201. https://doi.org/10. Methods in molecular biology. Springer, New York, 1038/nrd3052 pp 1–11 Skiles ML, Marzan AJ, Brown KS, Shamonki JM (2020) Wagner W, Ho AD (2007) Mesenchymal stem cell prepara- Comparison of umbilical cord tissue-derived mesenchymal tions—comparing apples and oranges. Stem Cell Rev stromal cells isolated from cryopreserved material and 3:239–248. https://doi.org/10.1007/s12015-007-9001-1 extracted by explantation and digestion methods utilizing a Wagner W, Wein F, Seckinger A et al (2005) Comparative split manufacturing model. Cytotherapy 22:581–591. characteristics of mesenchymal stem cells from human https://doi.org/10.1016/j.jcyt.2020.06.002 bone marrow, adipose tissue, and umbilical cord blood. Soler R, Orozco L, Munar A et al (2016) Final results of a phase Exp Hematol 33:1402–1416. https://doi.org/10.1016/j. I-II trial using ex vivo expanded autologous mesenchymal exphem.2005.07.003 stromal cells for the treatment of osteoarthritis of the knee Wang Q, Yang Q, Wang Z et al (2016) Comparative analysis of confirming safety and suggesting cartilage regeneration. human mesenchymal stem cells from fetal-bone marrow, Knee 23:647–654. https://doi.org/10.1016/j.knee.2015.08. adipose tissue, and Warton’s jelly as sources of cell 013 immunomodulatory therapy. Hum Vaccines Immunother Stolzing A, Jones E, McGonagle D et al (2008) Age-related 12:85–96. https://doi.org/10.1080/21645515.2015. changes in human bone marrow-derived mesenchymal 1030549 stem cells: consequences for cell therapies. Mech Ageing Zuk PA, Zhu M, Mizuno H et al (2001) Multilineage cells from Dev 129:163–73. https://doi.org/10.1016/j.mad.2007.12. human adipose tissue: implications for cell-based thera- 002 pies. Tissue Eng 7:211–228. https://doi.org/10.1089/ Thompson M, Mei SHJ, Wolfe D et al (2020) Cell therapy with 107632701300062859 intravascular administration of mesenchymal stromal cells continues to appear safe: an updated systematic review and Publisher’s Note Springer Nature remains neutral with meta-analysis. EClinicalMedicine. https://doi.org/10. regard to jurisdictional claims in published maps and 1016/j.eclinm.2019.100249 institutional affiliations. Vives J, Amposta J (2021) Risk management. In: Aljurf M, Snowden JA, Hayden P, Orchard KH, McGrath E (eds) 123