Immunologic Research https://doi.org/10.1007/s12026-023-09385-1 ORIGINAL ARTICLE Optimized reagents for immunopotency assays on mesenchymal stromal cells for clinical use Sílvia Torrents1,2 · Andrés Escudero del Moral1 · Margarita Codinach1,2 · Luciano Rodríguez1,2 · Sergi Querol1,2 · Joaquim Vives1,3,4 Received: 24 February 2023 / Accepted: 18 April 2023 © The Author(s) 2023 Abstract Multipotent mesenchymal stromal cells (MSC) offer new therapeutic opportunities based on their ability to modulate an imbal- anced immune system. Immunomodulatory potency is typically demonstrated in vitro by measuring the presence of surrogate markers (i.e., indoleamine-2,3-dioxygenase, IDO; tumor necrosis factor receptor type 1, TNFR1) and/or functional assays in co-cultures (i.e., inhibition of lymphoproliferation, polarization of macrophages). However, the biological variability of reagents used in the latter type of assays leads to unreliable and difficult to reproduce data therefore making cross-comparison between batches difficult, both at the intra- and inter-laboratory levels. Herein, we describe a set of experiments aiming at the definition and validation of reliable biological reagents as a first step towards standardization of a potency assay. This approach is based on the co-culture of Wharton’s jelly (WJ)-derived MSC and cryopreserved pooled peripheral blood mononuclear cells. Altogether, we successfully defined a robust and reproducible immunopotency assay based on previously described methods incorporating substantial improvements such as cryopreservation of multiple vials of pooled peripheral blood mononuclear cells (PBMC) from 5 individual donors that enable a number of tests with same reagents, also reducing waste of PBMC from individual donors and therefore contributing to a more efficient and ethical method to use substances of human origin (SoHO). The new methodology was successfully validated using 11 batches of clinical grade MSC,WJ. Methods described here contribute to minimize PBMC donor variability while reducing costs, streamlining assay setup and convenience and laying the foundations for harmonization of biological reagents usage in standardized immunopotency assays for MSC. Highlights • The use of pools of peripheral blood mononuclear cells (PBMCs) in potency assays contributes to robust and reproducible results, which is key in the assessment of mesenchymal stroma cells (MSC) potency for batch release. • Cryopreservation of PBMCs does not impact negatively on their activation and proliferation abilities. • Cryopreserved pools of PBMC constitutes convenient off-the-shelf reagents for potency assays. • Cryopreservation of pooled PBMCs from multiple donors is a way to reduce waste of donated PBMC and its associated costs, as well as reducing the impact of individual donor variability of substances of human origin (SoHO). Keywords Mesenchymal stromal cells · Immunomodulation · Quality & regulatory compliance · Batch release · Potency assay · Clinical use Background therapies, is hindered by the lack of harmonization of ana- lytical methods for determination of their critical quality Regulatory development of advanced therapy medicinal attributes (CQA), such as identity and potency [1, 2]. In products (ATMP), a new category of living medicines of addition to the lack of standardization, most potency assays complex biological nature including cell- and gene-based fail to reflect the mechanism of action (MoA) of the pro- posed treatment, as a result of poor understanding of actual biological processes involved in their therapeutic activity in * Joaquim Vives patients. In the case of multipotent mesenchymal stromal jvives@bst.cat cells (MSC), significant efforts have been made by scientific Extended author information available on the last page of the article 13 Vol.:(0123456789) Immunologic Research societies to propose criteria for their identification regard- 8.0% ± 10.4% in fresh and cryopreserved PBMC from three less of the tissue source [3–5]. However, developers tend to pools). In all cases, CFSE labelling was ≥ 85% efficient. adapt such criteria to their particular tissue sources of MSC, Considering that pooling 3 PBMC units was feasible, we manufacturing processes, cell characterization methods, also tested the possibility of using pools from 5 individual and estimated mechanisms of action, altogether resulting PBMC to generate more convenient volumes of reagents in different panels of CQA and specifications for MSC used suitable for use in a test laboratory. Therefore, the first series in clinical trials [6–9]. Consequently, this situation raises of pools tested were composed of 3 and 5 buffy coats from concerns on comparability of MSC from different labora- different donors (Fig. 1). Regardless of using individual or tories and even from different batches in the same produc- pooled PBMC preparations, proliferation was clearly stimu- tion facility, therefore jeopardizing the validity of systematic lated giving values of proliferating cells above 40% in all reviews and meta-analyses. In this context, efforts to pro- cases. Moreover, response of PBMC to polyclonal stimuli mote standardization would benefit the field by allowing for remained also above 40%, in all cryopreserved preparations more meaningful comparisons among studies, thus allowing (individual and pools) after thawing (Fig. 1). for a smoother clinical translation. It is therefore a pressing The mixed lymphocyte reaction (MLR) observed in need to define and standardise potency tests as a first step for PBMC pools is the result of an allogeneic immune reac- harmonization in the field [10]. Indeed replication as a result tion activating T cells that occurs when lymphocytes of dif- of standardization of methods and harmonization across lab- ferent donors are co-cultured without the need of external oratories would streamline research, gather relevant safety stimuli. In this sense, basal proliferation was lower in indi- and efficacy data as well as allowing for cost savings [11]. vidual PBMC preparations compared to pooled PBMNC, In the present study, we defined and qualified reagents and as expected (Fig. 1B’). Despite of MLR, basal proliferation then optimized methods for robust and reproducible immu- levels were significantly low (> 40% absolute proliferation in nopotency testing of MSC based on a previously described all cases) compared to the values achieved upon stimulation protocol by Oliver-Vila and collaborators [12], by incorpo- using strong activation stimuli (phorbol myristate acetate, rating substantial improvements such as cryopreservation of PMA; and ionomycin) in all cases, therefore providing an peripheral blood mononuclear cells (PBMC) pooled from ample range for the measurement of the inhibition of prolif- several donors to enable multiple tests with the same batch eration exerted by co-cultured MSC over background MLR of cellular reagents and therefore reducing the impact of (ranging from 46 to 65% and 46 to 72% absolute prolifera- individual donor variability on assay results. tion, in fresh and cryopreserved PBMC, respectively). Suitability of cryopreserved pools of PBMC in immunopotency testing of MSC Results As presented previously, MLR is responsible of increased basal The effect of cryopreservation on PBMC stimulation observed in pools compared to the use of individual donors (either fresh or cryopreserved). In order to determine We first evaluated the impact of cryopreservation on cellular whether MLR could interfere on the results of the potency viability, cell yield and proliferation capacity by comparing assays, we tested two independent MSC cell lines using PBMC individual and pool from 3 PBMC suspensions (9–13 mL from individual donors and pooled PBMC (Fig. 2). per donor). To do this, the feasibility of using pools was Remarkably, the use of pooled PBMC was found to be tested on (i) fresh and cryopreserved buffy coats, (ii) inves- suitable for use in this type of assays. The resulting values tigating efficiency of carboxyfluorescein succinimidyl ester of potency showed an acceptable range of variability, com- (CFSE) labelling in fresh and cryopreserved preparations prised either in the same range of those from individual (Fig. 1A), and (iii) evaluation of cell subpopulation com- donor PBMC for MSC line 1 (51% ± 3.8% vs. 50% ± 12%) position. Although the presence of lymphocytes was rela- or slightly higher when using pooled PBMC for MSC line tively similar in fresh and thawed cryopreserved paired 2 (73% ± 2.7% vs. 54% ± 12%) (Fig. 3). Importantly, in all samples, some differences were observed regarding mono- cases, cells were found in conformity with the release criteria cytes and granulocytes composition (Table 1). Despite using (> 30% inhibition of proliferation) established in the corre- cryopreservation medium containing dimethyl sulfoxide, a sponding Investigational Medicinal Product Dossier (IMPD) cryoprotective agent that preserves most attributes of fresh of the approved MSC,WJ-based ATMP. Moreover, improved samples, granulocytes were substantially reduced most comparable inter-assay results are offered, which is key for probably due to granule release and/or granulocyte death the validation of MSC batches, provided that sufficiently (10.6% ± 6.0% vs. 2.4% ± 0.5% in fresh and cryopreserved large volumes of cryopreserved PBMC may be used for the PBMC from three individual donors; and 20.8% ± 23.2% vs. assessment of immunopotency of different batches of MSC. 13 Immunologic Research Fig. 1  Proliferative capacity of cryopreserved individual and pooled tested fresh and after thawing paired cryopreserved PBMC prepara- PBMC preparations. Proliferative potential of PBMC (with or with- tions (B) Basal proliferation of individual PBMC and pools is high- out stimuli) was determined by CFSE staining and flow cytometry lighted in B’ box, showing higher proliferation in pools both in fresh analyses following the gating strategy shown in panel (A) Prolifera- and after thawing cryopreserved PBMC. Absolute proliferation high- tive potential of two individual (I1 and I2) and two pooled PBMC lights the operating range for immunopotency assays preparations (pools of 3 and 5 donors, P3 and P5 respectively) were Table 1  Comparison of composition and labelling efficiency in fresh and cryopreserved PBMC % CFSE labelling Lymphocytes Monocytes Granulocytes Fresh Cryo Fresh Cryo Fresh Cryo Fresh Cryo Individual PBMC 99.4 ± 0.4 99.9 ± 0.0 54.3 ± 3.1 57.4 ± 8.6 4.7 ± 2.2 6.3 ± 2.9 10.6 ± 6.0 2.4 ± 0.5 Pooled PBMC 99.5 ± 0.2 88.5 ± 0.5 57.4 ± 16.8 70.5 ± 7.9 10.6 ± 7.9 5.6 ± 5.3 20.8 ± 23.2 8.0 ± 10.4 Individual and pools of 3 PBMC preparations were analysed for composition (%) of main cell subpopulations as well as efficiency for carboxy- fluorescein succinimidyl ester (CFSE) labelling (%), expressed as mean ± standard deviation (n = 3). Please note that data presented is not from a donor-matched study Cryo cryopreserved 13 Immunologic Research 1–2 week each) in which PBMC stimulation and the inhi- bition of proliferation by MSC,WJ were assessed (Fig. 4). Reproducibility of our approach was assessed by thawing individual cryovials from the same PBMC pool and co- cultured with the same MSC line to evaluate the closeness of agreement between a series of measurements supporting inter-assay precision. Moreover, robustness was assessed by reproducing the method by different analysts (ST and AEdM) without the occurrence of unexpected differences in the obtained results. The inhibition of proliferation by MSC,WJ remained constant in tests performed at different times with cryo- preserved pools, also when compared to fresh PBMCs. Fig. 2  Inhibition of proliferation of two control MSC batches using Although variability was observed in terms of abso- individual and pooled PBMC preparations. Two independent lines lute proliferation (ranging from 65 to 75%), this had no of multipotent Mesenchymal Stromal Cells (MSC) were used for the assessment of immunopotency (expressed as percentage of inhibition impact on normalised values of inhibition of proliferation of the proliferation of labelled PBMC) using individual PBMC prepa- (Fig. 4). Indeed, variability in the results of the assay rations vs. a pool from three individual donors of PBMC (n = 3) was kept in a tight range comprised between 95 and 98% when using pools. In order to determine whether MLR remained stable with time, we studied proliferation of PBMC in basal and stimulation conditions of cells previously cryopreserved in aliquots from two independent pools of 5 donors, which were thawed and used in immunopotency assays at dif- ferent time points spanning more than 15 months (up to 399 days for pool A, and 468 days for pool B) (Fig. 3). For the two pools studied, the difference in the percentage of proliferating cells in the basal and stimulated conditions was very high for the first 12 months (average of 68%, ranging from 57 to 75%) and then reduced gradually at the two later time points (23% at 378 days and 19% at 399 days, respectively). Interestingly, despite the small differences (approximately 20%) of proliferating cells in the basal and stimulated conditions at later time points, we found that this range was sufficient to determine inhibi- tory properties of MSC,WJ on proliferation of PBMC in co-culture. Taken together the data presented previously, we pro- posed pools from 5 individual donors that, in our hands, resulted in a volume > 10 mL at 6–8 × ­107 cells/mL, result- ing in > 20 cryovials (composed of 0.5 mL of PBMC and 0.5 mL of cryopreservation solution). Although the inclu- sion of more donors would allow for a broader represen- tation of population variability and provide standardised Fig. 3  Stability of proliferation capacity of cryopreserved pools of reagents for conducting a larger number of assays, it is also PBMC. Cryovials from two pools of PBMC made from 5 donors each (in A, a pool composed of donor blood groups ­O+, ­A−, ­B+, ­A+, important to consider the burden of blood manipulation ­AB+; and in B, a pool composed of donor blood groups ­A+, ­O+, ­O+, and the need to adjust to the anticipated number of assays ­O+, A.+) were thawed at different times (up to 13 and 15 months, in a given period of time (one year in total at 1–2 assays respectively) and evaluated their capacity to proliferate in culture monthly, in our case) and stability (as shown in Fig. 3). with and without polyclonal stimuli. Pool in A showed high capacity to respond to proliferation stimuli, whereas pool in B showed lower Next, a control MSC line was used in four independ- capacity to respond to proliferation stimuli. All measurements were ent assays to validate repeatability and robustness, by performed on PBMC from cryovials thawed from either one of the conducting the assay at different time points (elapsed by two pools (A and B, respectively) 13 Immunologic Research Fig. 4  Proliferation of stimulated PBMC and inhibitory effect of ing with MSC,WJ was strong and showed a tight range between 95 MSC in co-culture. Although proliferation of stimulated PBMC and 98% in the three replicates performed (cryo 1, 2, and 3) and were ranged from 65 to 75%, inhibition of their proliferation by co-cultur- comparable to the values resulting from the fresh control (98%) Implementation of cryopreserved pools of PBMC donors. Attributes of MSC,WJ are described in Table 2. in routine release of MSC.WJ These data were compared to the results of tests per- formed using cryopreserved pooled PBMC from 5 dif- Finally, the success of the implementation of the ferent donors following the methodology described pre- improved methods for immunopotency assessment of viously. Interestingly we found no statistical differences MSC,WJ expanded from the same Master Cell Bank (P = 0.1858) between the two groups, showing simi- (same donor of umbilical cord) was evaluated using lar average values of inhibition of proliferation, being historical data of batches released from tests conducted 75% ± 18% (n = 9) vs. 69% ± 13% (n = 11), respectively, using fresh preparations of PBMC from individual while reducing variability of results (Fig. 5). Table 2  Identity and PBMC MSC batch CD45−/ CD31−CD73+ HLADR−/ CD90+ (%) Inhibition of immunopotency of clinical CD105+ (%) CD90+ proliferation bacthes of MSC,WJ (%) (%) Individual donors (fresh) 1 99.9 99.8 99.4 99.9 62% 2 99.9 99.9 99.3 99.7 97% 3 99.9 99.9 99.5 99.8 84% 4 100 99.9 99.5 99.7 103% 5 99.7 99.4 99.2 99.7 64% 6 99.9 99.7 99.5 99.9 69% 7 100 99.9 998 99.9 86% 8 99.7 99.8 99.8 99.9 57% 9 99.7 99.9 99.8 99.9 55% Pooled from 5 donors 10 99.8 99.9 99.9 100 86% (cryopreserved) 11 99.5 99.8 99.6 99.8 81% 12 99.1 99.8 99.9 100 70% 13 100 99.8 99.8 100 59% 14 100 99.5 99.6 100 61% 15 99.8 99.6 99.9 100 58% 16 99.4 99.7 99.6 100 73% 17 98.7 99.8 99.6 99.8 57% 18 98.7 99.6 99.6 100 63% 19 97.6 99.7 99.2 99.9 60% 20 99.8 99.6 99.4 100 65% 13 Immunologic Research potency assays prior to clinical testing, providing sound jus- tification of the scientific rationale and documented evidence [4, 17–20]. The establishment of suitable and reliable potency assays should guarantee batch-to-batch consistency for safe cellular products with the capacity to exert the intended thera- peutic effect [21]. Given both our poor understanding of MoA and the lack of relevant and validated animal models (which are also associated to high costs and ethical concerns on their use), in vitro assays are preferred. Indeed, MSC immunopo- tency potential is commonly assessed in cell-based assays using PBMC or ­CD3+ selected populations. Previously, we have proposed the use of fresh PBMC consisting of diverse cellular populations, mostly lympho- cytes but also monocytes and granulocytes, reproducing to some extent the complexity of cell types that are found in the patient [12]. Although our approach to cryopreserve PBMC has shown to alter the representation of the granulocytic population, this did not impact on the measurements of inhi- bition of overall cell proliferation, because proliferating cells are polyclonally stimulated lymphocytes. Given that contin- Fig. 5  Comparability of pooled vs. individual PBMC in the assess- uous improvements of potency assays must be done consist- ment of immunopotency of clinical batches of MSC,WJ drug prod- ently according to scientific and technological progress, we ucts derived from the same Master Cell Bank. Comparability study conducted on immunoptency assays of clinical-grade batches believe that improvements of the protocol presented herein MSC,WJ used in the context of ongoing Phase I/IIa trials (EudraCT will help to overcome existing limitations in the variability No.2021–000,346-18, Clinicaltrials.gov Id. NCT05054803; EudraCT of biological reagents for Quality Control (QC). No. 2020–001,505-22, ClinicalTrials.gov Id. NCT04390139; Importantly, we validated the new method in a clinically and EudraCT No. 2018–001,964-49, ClinicalTrials.gov Id. NCT03798353) and compassionate uses (in the management of relevant MSC-based ATMP. Particularly, we focused our graft versus host disease, GvHD). No statistically significant differ- efforts to standardize methods for scale up production of ences were observed between the potency assays performed on the multipotent MSC,WJ, as well as defining suitable QC assays group using individual donor of fresh PBMC (n = 9) and the group to determine identity, purity and potency consistently as a using cryopreserved pooled PBMC from 5 different donors (n = 11) (P = 0.1858; unpaired t test). Interestingly the range of inhibition val- pre-requisite for further clinical use in the context of inflam- ues was smaller showing lower variability in the group using pooled matory conditions, including spinal cord injury (EudraCT PBMC No. 2015–005,786-23, ClinicalTrials.gov Id. NCT03003364; and EudraCT No.2021–000,346-18, Clinicaltrials.gov Id. NCT05054803) and severe respiratory distress due to Discussion SARS-CoV-2 infection (EudraCT No. 2020–001,505-22, ClinicalTrials.gov Id. NCT04390139) [22–24]. Methods From all CQA commonly defined for MSC bioprocessing, for measuring potency of MSC,WJ relied initially on indi- those related to potency are mandatory for the release of clini- vidual blood donors for PBMC preparations but a pressing cally relevant batches as indicator of the product’s capacity need to reduce variability of biological reagents prompted to exert the desired therapeutic activity in patients [13, 14]. us to optimise the selection and their preparation. In addition According to the International Council for Harmonisation to their differentiation potential, MSC are of clinical inter- of Technical Requirements for Pharmaceuticals for Human est due to their ability to regulate inflammatory responses Use (ICH), potency is defined as the quantitative measure in diverse pathologies involving the immune system of the of biological activity based on attribute(s) of the product patient, being Graft versus Host Disease (GvHD) one of the linked to its relevant biological properties [15]. Adequate best studied conditions [25], although the mechanisms of potency assays are needed to predict the therapeutic efficacy action and the role that these cells play in immunomodula- of ATMPs, and these must be optimised and implemented tion are still an open question without conclusive answers. throughout the product development programme and not Altogether, our data demonstrates that pooled PBMCs are only after marketing approval [16]. However, this endeavour suitable for use in potency assays and may also contribute is challenging due to our limited understanding of actual MoA to the standardization of reagents for use in multiple assays in most clinical applications of MSC. Thus, both develop- intra- and/or inter-laboratory, therefore reducing the impact ers and regulators must agree on the choice of appropriate of variability of single-use reagents derived from different 13 Immunologic Research individual donors and thus making immunopotency assays Cell sourcing, MSC derivation, and expansion more robust and reproducible, while making the entire process more ethically conscious by reducing waste of the PBMC were obtained by a density gradient centrifugation (Histo- unused individual fresh PBMC. Moreover, our results pro- paque-1077; Sigma-Aldrich, Saint Louis, MO, USA) from 24- to vide evidence that cryopreserved MSC hold immunomodu- 48-h-old buffy-coats of healthy blood donors aged 18–70 years old, lation capacity after thawing contributing to an open dis- which were confirmed negative for serology markers: HBsAb, HIV cussion in the scientific literature in which some authors I/II, Lues (TPHA), Chagas, HBcAb, HCV, anti-HTLV I/II, NAD propose an additional passage to enhance MSC’s therapeutic (HCV-HIV, HBV). PBMCs were used individually or pooled, fresh potential [7, 26–28]. or cryopreserved, according to the experimental design. MSC were derived from the Wharton’s Jelly (WJ) of donated umbilical cords following GMP-compliant procedures reported elsewhere [29, 30], Conclusions with appropriate donor informed consent. After the isolation step, cells were replated by seeding T cell culture flasks at 1.5–3 × ­103 Cryopreserved pooled PBMC offer a reliable source of bio- cell/cm2. When the total number of viable cells reached at least logical reagents for consistent and reproducible assessment 5 × ­106, they were cryopreserved as master cell bank (MCB). Fur- of immunopotency by using an improved protocol of lym- ther expansion was performed after thawing vials of MCB using phoproliferation assay that was successfully validated using expansion medium composed of DMEM containing 2 mmol/L GMP-grade MSC,WJ. glutamine and supplemented with 10% pooled inactivated human serum B (hSerB; Banc de Sang i Teixits, Barcelona, Spain) [13, 31]. All cultures were maintained at 37 °C and 5% C­ O2 in humidi- Methods fied incubators. Media were replaced every 3–4 days, and trypsini- zation was performed at 70–90% confluence. Overall procedure details Cryopreservation The optimised protocol developed in this work is presented in a flowchart including sequential steps and in-process controls PBMC were collected from Ficoll gradient centrifugation (Fig. 6). Specific details are describe in the next subsections. of approximately 15 mL of buffy coat from each donor in TIME PROCESS DETAILS STEPS IN-PROCESS CONTROLS Ficoll gradient centrifugation in 1. PBMC isolation Cell count and viability assessment SepMateTM tubes DAY –N Cryopreservation 30E06 pooled PBMCs per cryovial 2. PBMC cryopreservation PBMC cryopreservation batch validation Thawing of MSC (optional) 3. Establishment of the Cell count and viability assessment DAY -6±2 immunomodulatory cell population (MSC) Expansion of MSC Seeding of MSC on 24-well plates 4. MSC seeding Cell count and viability assessment (≈ 5E04 cells/cm 2) 5. PBMC thawing Cell count and viability assessment DAY -1 CFSE 0.625-2 µM in DPBS 5E06 PBMC/mL Cell count and viability assessment post- 6. PBMC CFSE staining 20 min, dark, RT labeling reaction labeling reaction and after overnight Overnight, dark, 37ºC stabilization period with expansion medium 1E06 PBMC/mL DAY 0 1:5 MSC : PBMC 7. CFSE-PBMC seeding with stimulation Adherence of MSC to plastic media 0.250 µg/Ml PMA + 0.5 µM ionomycin 8. Proliferation analysis by flow cytometry Proliferating PBMC DAY +5 Fig. 6  Flowchart. Sequential steps, time considerations, and in-process controls 13 Immunologic Research Sepmate tubes (StemCell Technologies, Vancouver, CA). 5:1 PBMC:MSC,WJ ratio. Typically, MSC,WJ cultures required Next, pools were prepared and cells were cryopreserved in a 1–2 passages upon thawing GMP grade cryovials. Lymphocytes solution composed of Dulbecco’s Phosphate-Buffered Saline were activated with 25 ng/mL Phorbol 12-myristate 13-ace- (DPBS; Gibco) supplemented with 10% (v/v) dimethyl sul- tate (PMA, Sigma-Aldrich) and 0.5 μM Ionomycin (Sigma- foxide (DMSO; OriGen Biomedical, Austin, TX, USA) and Aldrich) in a final volume of 0.5 mL/well of DMEM containing 2% (w/v) human serum albumin (HSA; Grifols, Barcelona, 2 mmol/L glutamine and supplemented with 10% hSerB. Prolif- Spain), by applying a controlled freezing rate of 1 °C/min in eration of PBMC was determined by measuring the reduction of a Mr. Frosty device (Nalgene, Rochester, NY, USA) kept in fluorescence intensity at day 5 by flow cytometry using a Navios a − 80 °C freezer for 24 h before storage at − 196 °C in a liquid EX, and raw data were analyzed with Kaluza Analysis Software nitrogen tank until further use [32]. When needed, typically (version 2.1, Beckman Coulter). Inhibition of PBMC prolifera- within 28 days at the times shown in Fig. 3) cells were rapidly tion was calculated using Eqs. 1, 2 and 3, as described next: thawed in a 37 °C water bath, then slowly diluted 1:10 using pre-cooled thawing solution consisting of 10% (w/v) albumin absolute proliferation = stimulated − nonstimulated (1) in Plasmalyte 148. DMSO was washout by centrifugation at 340 g for 10 min and the cell pellet was resuspended in appro- absolute proliferation w∕ MSC normalized proliferation (%) = x 100 (2) priate culture medium according to the experimental design. absolute proliferation w∕o MSC Flow cytometry Inhibitition (%) = 100 − normalized proliferation of co − culture (3) Cells were enumerated using Perfect-Count Microspheres™ (Cytognos, Salamanca, Spain) microbeads in a Navios EX flow Statistical analysis cytometer (Beckman Coulter, Pasadena, CA, USA). Viabil- ity (%) was determined using the 7-Amino-Actinomycin D Descriptive data were expressed as mean ± standard devia- (7-AAD, Beckman Coulter, Pasadena, CA, USA) exclusion tion (number) or range, as detailed in the text. Statistical method. Data were analyzed with Navios EX Software v2.0 significance was assessed by the t-test as described in the (Beckman Coulter) software. In accordance with de Interna- text and figure legends using GraphPad Prism (GraphPad tional Society on Cell and Gene Therapy (ISCT) criteria [3], software Inc., La Jolla, CA). identity of MSC was evaluated by the expression of surface markers CD31 PacificBlue-conjugated (clone 5.6E; Beckman Acknowledgements The authors are grateful to Clémentine Mirabel, Coulter), CD45 KromeOrange-conjugated (clone J33; Beckman analysts from Laboratori Cel·lular at BST, Margarita Blanco and Dr. Gemma Aran for technical support. Coulter), CD73 PE-conjugated (clone AD-2; Beckman Coulter), CD90 FITC-conjugated (clone F15-42–1-5, Beckman Coulter), Author contribution ST and JV conceived the study and prepared the CD105 PC7-conjugated (clone TEA3/17.1.1; Beckman Coul- first draft; ST and AEdM performed experiments, analyzed data, and ter), and HLA-DR APC-conjugated (clone Immu-357; Beckman revised the manuscript; MC, LR, and SQ revised data and revised the manuscript. All authors read and approved the final manuscript. Coulter) in a Navios EX Device. Cells were stained for 15 min at room temperature, washed and resuspendend with DPBS. Funding Open Access Funding provided by Universitat Autonoma de Acquisition was done using Navios EX, and raw data were ana- Barcelona. This work has been developed in the context of Red Espa- lyzed with Navios EX Software (version 2.0, Beckman Coulter). ñola de Terapias Avanzadas (TERAV, expedient no. RD21/0017/0022) funded by Instituto de Salud Carlos III (ISCIII) in the context of Next- GenerationEU’s Recovery, Transformation and Resilience Plan and by Lymphocyte proliferation assay the Commission for Universities and Research of the Department of Innovation, Universities, and Enterprise of the Generalitat de Catalunya The immunomodulation potential of MSC,WJ was determined (2017 SGR 719). by their capacity to inhibit the proliferation of polyclonally Data availability All data generated or analyzed during this study are stimulated lymphocytes in vitro, following an improved included in this published article and its additional file. method based on that originally described by Oliver-Vila and collaborators [12], as described next. Briefly, 2.5 × ­106 PBMC/ Declarations mL were labelled with 0.625 μM carboxy–fluorescein diac- Ethics approval and consent to participate Cells were sourced from Banc etate succinimidyl ester (CFSE) for 20 min at 37ºC using the de Sang i Teixits’ Biobank (Barcelona, Spain) and had appropriate donor CellTrace™ CFSE Cell Proliferation Kit (Molecular Probes, informed consent for use in research. Authorisation issued by Hospital de Eugene, OR, USA). After washing, (1, 2) × ­107 cells/mL were la Vall d’Hebron’s Ethics Committee (Barcelona, Spain) to JV. incubated for 12 min at 37 °C, washed again and seeded onto Consent for publication Not applicable. flat-bottomed 24-well plates (Corning, Corning, NY, USA) at 13 Immunologic Research Competing interests The authors declare no competing interests. assessment of immunomodulative potential of clinical grade multipo- tent mesenchymal stromal cells. Cytotechnology. 2018;70(1):31–44. 13. Grau-Vorster M, Rodriguez L, Del Mazo-Barbara A, Mirabel C, Open Access This article is licensed under a Creative Commons Attri- Blanco M, Codinach M, et al. Compliance with good manufac- bution 4.0 International License, which permits use, sharing, adapta- turing practice in the assessment of immunomodulation poten- tion, distribution and reproduction in any medium or format, as long tial of clinical grade multipotent mesenchymal stromal cells as you give appropriate credit to the original author(s) and the source, derived from Wharton’s jelly. Cells. 2019;8(5):484. https://​doi.​ provide a link to the Creative Commons licence, and indicate if changes org/​10.​3390/​cells​80504​84. were made. The images or other third party material in this article are 14. CHMP. ICH guideline Q8 (R2) on pharmaceutical development step included in the article's Creative Commons licence, unless indicated 5. In: Products CfHM, editor. 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Authors and Affiliations Sílvia Torrents1,2 · Andrés Escudero del Moral1 · Margarita Codinach1,2 · Luciano Rodríguez1,2 · Sergi Querol1,2 · Joaquim Vives1,3,4 2 Sílvia Torrents Transfusion Medicine Group, Vall d’Hebron Research storrents@bst.cat Institute, Universitat Autònoma de Barcelona, Passeig de La Vall d’Hebron 129‑139, 08035 Barcelona, Spain Andrés Escudero del Moral 3 andresescuderom@iqs.edu Musculoskeletal Tissue Engineering Group, Vall d’Hebron Research Institute (VHIR), Universitat Autònoma Margarita Codinach de Barcelona, Passeig de La Vall d’Hebron 129‑139, mcodinach@bst.cat 08035 Barcelona, Spain Luciano Rodríguez 4 Departament de Medicina, Universitat Autònoma de lrodriguez@bst.cat Barcelona, Passeig de La Vall d’Hebron 129‑139, Sergi Querol 08035 Barcelona, Spain squerol@bst.cat 1 Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jordà, Passeig Taulat, 116, 08005 Barcelona, Spain 13