EBioMedicine 54 (2020) 102729 Contents lists available at ScienceDirect EBioMedicine journal homepage: www.elsevier.com/locate/ebiom Research paper First-in-human PeriCord cardiac bioimplant: Scalability and GMP manufacturing of an allogeneic engineered tissue graft Cristina Prat-Vidala,b,c,d, Luciano Rodríguez-Go meze, Miriam Aylagase,f, Nuria Nieto-Nicolaug, Paloma Gastelurrutia a,b,c,d g , Elba Agustí , Carolina Galvez-Monto na,b,c, Ignasi Jorbah, b Albert Teis , Marta Monguio -Tortajada , Santiago Roura a,i a,b,c , Joaquim Vivese,j,k, Silvia Torrents-Zapatae, María Isabel Cocae, Laura Realese, María Luisa Ca mara-Rosellb, Germa n Cediel , Ruth Coll , Ramon Farre b l  , Daniel Navajas , Anna Vilarrodonag, m h Joan García-Lopez , Christian Mun l ~ oz-Guijosab, Sergi Querole,f, Antoni Bayes-Genisa,b,c,k,* a ICREC Research Program, Germans Trias i Pujol Health Science Research Institute, Can Ruti Campus, Badalona, Spain b Heart Institute (iCor), Germans Trias i Pujol University Hospital, Carretera de Canyet s/n, 08916 Badalona, Spain c CIBER Cardiovascular, Instituto de Salud Carlos III, Madrid, Spain d  Biomedica de Bellvitge - IDIBELL, L'Hospitalet de Llobregat, Spain Institut d'Investigacio e Cell Therapy Service, Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jorda, Passeig Taulat, 116, 08005 Barcelona, Spain f Transfusional Medicine Group, Vall d'Hebron Research Institute, Autonomous University of Barcelona, Barcelona, Spain g Barcelona Tissue Bank (BTB), Banc de Sang i Teixits (BST), Barcelona, Spain h Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology, CIBERES, University of Barcelona, Barcelona, Spain i REMAR-IVECAT Group, Germans Trias i Pujol Health Science Research Institute, Can Ruti Campus, Badalona, Spain j Musculoskeletal Tissue Engineering Group, Vall d'Hebron Research Institute (VHIR), Autonomous University of Barcelona, Passeig de la Vall d’Hebron 129-139, 08035 Barcelona, Spain k Departament de Medicina, Universitat Auto noma de Barcelona, Passeig de la Vall d’Hebron 129-139, 08035 Barcelona, Spain l Research and Education. Banc de Sang i Teixits, Edifici Dr. Frederic Duran i Jorda , Passeig Taulat, 116, 08005 Barcelona, Spain m Unit of Biophysics and Bioengineering, School of Medicine and Health Sciences, University of Barcelona-IDIBAPS-CIBERES, Barcelona, Spain A R T I C L E I N F O A B S T R A C T Article History: Background: Small cardiac tissue engineering constructs show promise for limiting post-infarct sequelae in Received 17 December 2019 animal models. This study sought to scale-up a 2-cm2 preclinical construct into a human-size advanced ther- Revised 24 February 2020 apy medicinal product (ATMP; PeriCord), and to test it in a first-in-human implantation. Accepted 5 March 2020 Methods: The PeriCord is a clinical-size (12 16 cm2) decellularised pericardial matrix colonised with human Available online xxx viable Wharton’s jelly-derived mesenchymal stromal cells (WJ-MSCs). WJ-MSCs expanded following good manufacturing practices (GMP) met safety and quality standards regarding the number of cumulative popu- Keywords: lation doublings, genomic stability, and sterility. Human decellularised pericardial scaffolds were tested for Wharton’s jelly-derived mesenchymal stromal cells (WJ-MSCs) DNA content, matrix stiffness, pore size, and absence of microbiological growth. Biofabrication Findings: PeriCord implantation was surgically performed on a large non-revascularisable scar in the inferior Advanced therapy medicinal product (ATMP) wall of a 63-year-old male patient. Coronary artery bypass grafting was concomitantly performed in the Cardiac tissue engineering non-infarcted area. At implantation, the 16-cm2 pericardial scaffold contained 12¢5 £ 106 viable WJ-MSCs Scaffold (85¢4% cell viability; <0¢51 endotoxin units (EU)/mL). Intraoperative PeriCord delivery was expeditious, and Myocardial infarction secured with surgical glue. The post-operative course showed non-adverse reaction to the PeriCord, without requiring host immunosuppression. The three-month clinical follow-up was uneventful, and three-month cardiac magnetic resonance imaging showed ~9% reduction in scar mass in the treated area. Interpretation: This preliminary report describes the development of a scalable clinical-size allogeneic Peri- Cord cardiac bioimplant, and its first-in-human implantation. Funding: La Marato  de TV3 Foundation, Government of Catalonia, Catalan Society of Cardiology, “La Caixa” Banking Foundation, Spanish Ministry of Science, Innovation and Universities, Institute of Health Carlos III, and the European Regional Development Fund. © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/) * Corresponding author at: Heart Institute (iCor), Germans Trias i Pujol University Hospital, Carretera de Canyet s/n, 08916 Badalona (Barcelona), Spain. E-mail address: abayesgenis@gmail.com (A. Bayes-Genis). https://doi.org/10.1016/j.ebiom.2020.102729 2352-3964/© 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/) 2 C. Prat-Vidal et al. / EBioMedicine 54 (2020) 102729 allogeneic investigational advanced therapy medicinal product (ATMP; named PeriCord). We also report preliminary safety data Research in context from the first PeriCord implantation performed in a roll-in patient from the ongoing phase I PERISCOPE (the PERIcardial matrix with Evidence before this study mesenchymal Stem Cells fOr the treatment of PatiEnts with infarcted myocardial tissue) clinical trial (EudraCT N°. 2018-001964-49; Clini- MI sequelae remain a major cause of morbidity and mortality. calTrials.gov identifier: NCT03798353). Small-sized cardiac tissue engineered grafts proved safe and effective in the MI swine model. 2. Materials and methods Added value of this study 2.1. Rational of the study Here we developed and performed the first-in-human surgery of a scalable clinical-size PeriCord cardiac bioimplant. The main objective of this study was to scale up a small-size pre- clinical construct into a human-size allogeneic investigational ATMP, Implications of all the available evidence named PeriCord, following GMP-compliant procedures and to test it The PeriCord, composed of human WJ-MSCs within decellular- in a first-in-human implantation in the context of the PERISCOPE ised pericardium, was feasible and preliminarily safe, without clinical trial (Fig. 1). This novel ATMP combines WJ-MSCs as active requirement of host immunosuppression after implantation. ingredient and human decellularised, lyophilised, and sterilised peri- Long-term follow up and completion of the PERISCOPE trial cardium, acting as a cell supportive material (vehicle) for surgical will confirm the value of this first-in-class PeriCord cardiac implantation. bioimplant. Umbilical cord (UC) samples were collected within the Concordia Program (http://www.bancsang.net/professionals/en_concordia) with appropriate donor informed consent. UC-mesenchymal stromal cells (MSCs) were derived from the Wharton’s jelly (WJ) in qualified areas of Blood and Tissue Bank of Catalonia (BST, Barcelona), following GMP- 1. Introduction compliant procedures. Human pericardium samples were obtained from Myocardial infarction (MI) causes irreversible cardiac muscle loss, non-contractile scar formation, and, ultimately, a maladaptive adverse ventricular remodelling resulting in end-stage ischemic heart failure [1]. Over recent decades, many small-scale clinical trials have tested cell-based therapies using distinct cell sources, with the aim of regenerating infarcted hearts [2 5]. Many of these trials have reported only modest improvements in cardiac function, which are unlikely to be clinically relevant [6]. Main sources of mesenchymal stromal cells (MSCs) include bone marrow, adipose tissue, and Wharton’s jelly from the umbilical cord discarded after birth. Notably, large quantities of allogeneic MSCs can be most easily collected from Wharton’s jelly, and these Wharton’s jelly-derived MSCs (WJ-MSCs) are more primitive than MSCs derived from more mature tissue sources, exhibiting intermediate properties between those of embryonic and adult stem cells [7]. Moreover, WJ- MSCs have a lower risk of carrying somatic mutations, extensive expansion capacity in culture, immunological immaturity, and immu- nomodulatory properties, making them optimal candidates for allo- geneic cell therapy. Nevertheless, the mechanisms of action of MSCs are not fully deciphered. MSCs are associated with regenerative and immunomodulatory capabilities, including monocyte modulation toward an anti-inflammatory phenotype that facilitates tissue repair [8,9]. We observed that WJ-MSCs polarise monocytes towards a regu- latory M2 phenotype, and strongly inhibit inflammatory responses of stimulated allogeneic T cells [10,11]. Cardiac tissue engineering is a therapeutic strategy that involves delivery of both cells and supportive biomaterials into injured myo- cardium, thus improving cell retention and survival compared with direct cell injection delivery [12,13]. We previously demonstrated that a small (2 cm2) engineered preclinical cardiac graft comprising human decellularised pericardium and MSCs was safe and effective in an MI porcine model [14 16]. Once implanted over the ischemic myocardium, the graft became vascularised and innervated, promot- ing damaged tissue revascularisation, reducing infarct size, amelio- rating the adverse remodelling process and fibrosis progression, and ultimately improving cardiac function [14 16]. In the present study, we describe the translational process to the Fig. 1. Schematic of the two components of the PeriCord bioimplant, human WJ-MSCs clinic, including graft scalability and good manufacturing practice and human decellularised pericardium. The PeriCord was safely implanted in a first- (GMP) biofabrication, from a small-size preclinical construct to an in-human patient. C. Prat-Vidal et al. / EBioMedicine 54 (2020) 102729 3 deceased donors and personal information were treated in accordance analysed with BD CellQuestTM Pro-software (version 5.2.1.; BD Bioscien- with European Tissue and Cell Directives (2004/23/CE, 2006/17/CE, and ces, Franklin Lakes, NJ, USA). WJ-MSC potency was evaluated using 2006/86/CE), and with the local legal requirements and the Laws for the peripheral blood mononuclear cells (PBMCs) from healthy blood donors Development and Applications of Organ Transplants. Informed consent (BST), obtained by density gradient (Histopaque-1077; Sigma-Aldrich was obtained for clinical application and/or applied research. Inc., St. Louis, MO, USA). To assess cell proliferation, isolated PBMCs With respect to active ingredient, we obtained n = 4 batches of clin- were labelled with carboxy-fluorescein diacetate succinimidyl ester ical-grade WJ-MSCs. In all cases, serological, microbiological, and (CFSE; Invitrogen, Carlsbad, CA, USA), as previously described [19]. adventitious virus testing, karyotyping, sterility, Mycoplasma, endo- The clinical-grade WJ-MSCs met several safety and quality stand- toxin levels, phenotypic profiling and immunomodulatory assay, were ards with regards to number of cumulative population doublings, performed throughout manufacturing process in order to confirm WJ- genomic stability, and sterility, which were examined according to MSCs’ identity and potency, to ensure batch-to-batch bioequivalence, the investigational medicinal product dossier approved by the Span- and to guarantee suitability for clinical use. Viability of initial thawed ish Agency of Medicines and Medical Devices (AEMPS; PEI16-017). WJ-MSCs (n = 37), following sucrose resuspension (n = 37) and after Table 1 summarises the acceptance criteria for all the in-process con- 16 h at 2 8 °C (n = 20) were also analysed by flow cytometry. trols during clinical-grade WJ-MSCs manufacturing. Concerning vehicle component, a total of n = 35 human pericardia were cut and decellularised. Different square pieces of ~16 cm2 and 2.2.2. Vehicle: decellularised, lyophilised and sterilised human ~1 cm2 obtained from each pericardium were randomly assigned to pericardium different experimental sets, as follows: n = 5 pieces, to measure the Human pericardium samples (n = 35) were obtained from effect of decellularisation on pericardial matrix dimensions; n = 4 pairs deceased donors within 24 h post-death, and frozen at 80 °C. Each of both sizes, to quantify DNA residual content after decellularisation; pericardium was cut into square pieces of 4 £ 4 cm (~16 cm2) and n = 4 pieces, for histological and immunohistochemical analysis; n = 4 1 £ 1 cm (~1 cm2). pairs of both sizes, for mechanical testing; n = 11 pieces, to analyse Decellularisation in the BST-qualified areas was performed as previ- pore size and roundness ratio in decellularised pericardial matrices; ously reported by our group [14], with some modifications compatible and n = 4 pairs of both sizes, for validation of the final decellularisation with the quality-by-design approach to ensure human tissue quality protocol. Upon validation of decellularisation, n = 3 pericardia were (see Supplementary Materials). The procedure was validated using two used to validate manufacturing procedure of the vehicle (final matrix), paired quadrangular pieces of pericardium (n = 4 pairs) to demonstrate including serological and microbiological testing, sterility assessment, equivalence between both sizes. Acceptance criteria for each analysed final quadrangular size, residual moisture, and citotoxicity testing. parameter in decellularised pericardia based on the former preclinical During the scale-up process, n = 37 matrix colonisations were per- construct properties, included DNA content, matrix stiffness, pore size, formed using post-thawed sucrose-conditioned WJ-MSCs. After rieux, Inc., and microbiological growth (BACT/ALERTÒ method; BioMe sequential cell seeding, WJ-MSCs viability was verified in samples of Durham, NC, USA) (Supplementary Table 1). both ~16 cm2 and ~1 cm2 from n = 3 vehicle batches. Following the Each vehicle batch contained two fragments (~16 cm2 and ~1 cm2) of defined manufacturing protocol, WJ-MSCs’ colonisation efficiency human decellularised, lyophilised, and sterilised pericardium. The (n = 47), dose range (n = 47), and viability (n = 27) were determined ~16 cm2 was used for PeriCord manufacturing, and the ~1 cm2 for an in- in the finished product (FP). Finally, PeriCord’s manufacturing process process quality control (QC; cell viability assessment). Vehicle batch (n = 4), stability (n = 3) and transportation (n = 3) were also validated release criteria included negative serological (same as indicated in at room temperature (RT) before the first PeriCord manufactured to Table 1 for umbilical cord blood) and microbiological testing of the be implanted in a roll-in patient of the PERISCOPE trial. 2.2. Investigational medicinal product: the PeriCord Table 1 Acceptance criteria for the manufacturing of clinical-grade WJ-MSCs. 2.2.1. Active ingredient: clinical-grade WJ-MSCs WJ-MSCs were derived from specifically donated umbilical cord In-process Acceptance Manufacturing quality control criteria process tissue following previously described GMP-compliant procedures [17] in the classified areas of the Blood and Tissue Bank of Catalonia UC blood Serology: HBsAg, HIV I/II, Lues Negative (BST; Barcelona, Spain). Briefly, clinical-grade WJ-MSCs were (TPHA), Chagas, anti-HBc, HCV, anti-HTLV I/II, NAT obtained using a two-tiered cell banking system comprising an initial (HCV-HIV, HBV) master cell bank (MCB), and a second cell bank, named drug product WJ-MSC expansion Dose 2¢5 £ 107 § 20% (DP), containing ready-to-use pure WJ-MSCs. Immediately after cell step and scale-up viable cells/cryotube expansion, DP was cryopreserved in units containing 2¢5 £ 107 § 20% to DP Viability 70% cells/cryovial in PlasmalyteÒ 148 (Baxter, Deerfield, IL, USA) supple- Karyotype Non-chromosomal abnormalities mented with 4% w/v human serum albumin (Grifols, Barcelona, CD105+/CD45 (%) 95% Spain) and 10% v/v dimethyl sulfoxide (CryoservÒ , Bioniche Pharma, CD73+/CD31 (%) 95% Lake Forest, IL, USA), and stored until PeriCord manufacturing. CD90+ (%) 95% All DP batches were assayed for identity (phenotype) and potency HLA-DR Informative* Mycoplasma Negative (immunomodulatory potential) to ensure bioequivalence between Endotoxin 1 EU/mL batches. Phenotype was evaluated by flow cytometry. WJ-MSCs were Sterility Sterile immunostained with specific antibodies against the surface markers Adventitious virus Negative CD31, CD45, CD73, CD90, CD105, and HLA-DR, based on the mesenchy- Immunomodulation >30% Inhibition mal identity criteria established by the International Society for Cellular (Potency assay) of PBMC proliferation Therapy (details in Supplementary Materials) [18]. Cell number and via- Note: DP, drug product; EU, endotoxin units; HBsAg, hepatitis B surface antigen; bility were determined by Perfect-Count MicrospheresTM (Cytognos, HBc, hepatitis B core antigen; HBV, hepatitis B virus; HCV, hepatitis C virus; HTLV, human T-cell leukemia-lymphoma virus; HIV, human immunodeficiency virus; Salamanca, Spain) microbeads and 7-amino-actinomycin D (BD Bio- NAT, nucleic acid test; PBMC, peripheral blood mononuclear cell; TPHA, Treponema sciences, Franklin Lakes, NJ, USA) exclusion method, respectively, fol- pallidum hemagglutination assay; UC, umbilical cord; WJ-MSCs, Wharton’s jelly lowing manufacturer’s instructions. Acquisition was performed using a mesenchymal stromal cells. FACSCaliburTM flow cytometer (BD, Franklin Lakes, NJ, USA), and data * HLA-DR for informative purposes only. 4 C. Prat-Vidal et al. / EBioMedicine 54 (2020) 102729 pericardial donors, no evidence of microbial growth with the BACTECTM consisting in a sequential cell seeding by sectors onto the vehicle was system (BD, Franklin Lakes, NJ, USA), final regular quadrangular size of chosen. Then, PeriCord was an investigational ATMP defined as a 12 16 cm2, homogeneous density, lack of orifices, residual moisture human decellularised, lyophilised, and sterilised pericardial matrix <10%, and gamma irradiation (25 35 kGy). (12 16 cm2) colonised with viable WJ-MSCs in 0.15% v/v PuraStatÒ (3-D Matrix Inc., Tokyo, Japan) and 10% w/v sucrose (Sigma-Aldrich 2.2.3. PeriCord manufacturing Inc., St. Louis, MO, USA). PeriCord was manufactured at GMP-certified The development and design of PeriCord (FP) had our preclinical facilities (BST), following AEMPS-approved procedures (PEI18-140). experience as starting point. Different colonisation strategies were PeriCord biofabrication involved multiple steps and in-process con- assayed in order to replicate the composition of the preclinical con- trols performed on the same day as FP batch release for clinical use struct (2 cm2) in a larger scale (12 16 cm2). A final protocol (Fig. 2). The main steps were vehicle immobilisation in the primary Fig. 2. Schematic of clinical-grade PeriCord manufacturing process, and established in-process QCs. C. Prat-Vidal et al. / EBioMedicine 54 (2020) 102729 5 packaging, WJ-MSCs thawing (2 units of DP), cell conditioning and state free-precession sequence [repetition time 44¢70 ms; echo time dose adjustment, vehicle colonisation, and final packaging in a tem- 1¢26 ms; flip angle 78°; matrix 272; spatial resolution (1¢3 1¢5) x perature-monitored triple-container system with PlasmalyteÒ 148 (1¢3 1¢5) x 8 mm, depending on the field of view] for cine imaging in solution for PeriCord transportation to the hospital before implanta- parallel short-axis (contiguous slices of 8-mm thickness covering tion (details in Supplementary Materials). A small-scale (~1 cm2) rep- from base to apex), and three long-axis views of the left ventricle. lica of each FP was proportionately manufactured in parallel, for in- Delayed enhancement images were acquired with a segmented gra- process cell viability assessment. We also performed validation of the dient-echo inversion-recovery sequence [repetition time scale-up procedure, PeriCord manufacturing process, in-process QCs, 600 800 ms, depending on the cardiac heart rate; echo time and FP stability and transportation. In-process QC strategy in the FP 3¢24 ms; flip angle 25°; matrix 256; spatial resolution include: WJ-MSCs dose calculation, based on cell count method; the 1¢3 £ 1¢3 £ 8 mm] at matching cine-image slice locations 10 to microbiological control, endotoxin levels, and Mycoplasma test on 20 min after intravenous gadolinium-DTPA administration washing supernatants (as previously described for clinical grade WJ- (0¢2 mmol/kg; Gadovist, Bayer Schering Pharma AG, Berlin, Germany) MSCs batch release); environmental controls (particles and microbi- [20]. We optimised the inversion time to null the normal myocar- ology); and the viability of cells colonising matrix, determined by dium and adjusted views per segment, and trigger delay according to LIVE/DEADTM method in the small-scale replica (Fig. 2).The accep- the patient’s heart rate. tance criteria for initial PeriCord batch certification comprised a dose All images were reviewed and analysed off-line with a specialised range of (7 15) £ 106 total viable WJ-MSCs, 70% viability and endo- post-processing software (IntelliSpace Portal, Philips, Eindhoven, NL) toxin levels 4 endotoxin units (EU)/mL. by an independent operator (A.T.). Left ventricular (LV) endocardial PeriCord stability was determined by analysing viability of WJ- border (papillary muscles were excluded) were manually traced on MSCs colonising matrix with LIVE/DEADTM method at different time all short-axis cine images at the end-diastolic and end-systolic frames points (from 0 to 96 h), maintaining the FP in the final packaging at to determine the LV end-diastolic and LV end-systolic volumes, RT (n = 3). PeriCord transportation was also validated to evaluate the respectively. LV mass was calculated by subtracting the endocardial impact of shipment on the final cell dose. Thus, FP and corresponding volume from the epicardial volume at end diastole and then multi- small-replica (n = 3 each) were shipped from BST to the Germans plying by the tissue density (1¢05 g/mL) [21,22]. Trias i Pujol University Hospital (12-km distance) at RT in a tempera- The endocardial and epicardial contours on delayed enhancement ture-monitored container, in accordance with P650 Packaging Regu- images were also outlined manually. Regions of interest (ROIs) were lations of the European Agreement Concerning the International then manually traced in the hyper-enhanced area at place of maxi- Carriage of Dangerous Goods by Road (ADR) for the transport of bio- mum signal intensity, and in the normal-appearing remote myocar- logical substances, category B (Class UN 3373). dium. As previously described, the areas of hyper-enhancing myocardium were then automatically segmented by using a full- 2.3. PeriCord first-in-human surgical implantation width at half-maximum (FWHM) algorithm [23,24]. Two corrections were required for all automated ROIs. First, microvascular obstruction Here we report preliminary data for up to three months following (defined as hypointensity within a hyperintense region in patients the PeriCord implantation in a roll-in patient from the PERISCOPE with infarctions) was adjusted to be included as late gadolinium trial. The PERISCOPE trial is a first-in-human, phase I, double-blind enhancement if present. Second, any obvious blood pool or pericar- (patients and clinical cardiologist), single-centre clinical trial dial partial volume and artefacts were further removed from the ROI. (NCT03798353). This study was approved by the AEMPS and the local Scar volume for each slice was calculated as: area scar £ slice Ethics Committee from the Germans Trias i Pujol University Hospital thickness. The scar mass was expressed as total scar volume £ 1¢05 g. (Eudra CT No. 2018-001964-49) and follows the ethical precepts of The scar percentage of myocardium was also expressed as a percent- the Declaration of Helsinki and the Guidelines on Good Clinical Prac- age of the total myocardial volume: volume scar/volume tice specific to ATMPs. The presently reported patient gave signed myocardium £ 100. informed consent prior to enrolment. The trial will enrol a total of 12 patients with non-revascularisable 2.5. Statistical analysis myocardial scars who are candidates for surgical revascularisation of other myocardial areas, including two roll-in open-labelled patients, Data are presented as mean § SEM. Statistical analyses for cyto- and ten randomised patients to a control or treatment group (1:1 toxicity experiments were performed using the independent Stu- ratio, using a computer-generated sequence). Following inclusion in dent’s t-test. The paired sample t-test was used to analyse vehicle the study (first visit), control patients will undergo coronary artery area reduction and vehicle size comparisons (DNA content, pore size, bypass graft (CABG) for the revascularisable region(s). Treatment and roundness in 16 cm2 vs. 1 cm2). One-way ANOVA with Tukey’s patients will undergo the required CABGs and also receive PeriCord post-hoc correction for multiple comparisons were used for DNA applied by surgical glue (GlubranÒ 2, CardioLink, Barcelona, Spain) content, pore size, and mechanical data. Non-parametric Friedman over the non-revascularisable myocardial scar. Patients will have fol- and Wilcoxon Signed-Rank tests were used to assess differences in low-up visits at one-week post hospital discharge, three, six, and 12 cell viability by sequential colonisation strategy and vehicle size, months. The primary endpoint is safety, according to a combination respectively. Data were analysed using SPSS 21.0.0.0 (SPSS Inc., Chi- of clinical severe events (death or all-cause rehospitalisation) and cago, IL, USA), GraphPad Prism 6 (GraphPad Software, San Diego, CA, severe adverse reaction to the PeriCord. Safety data will be assessed USA), and SigmaPlot 13 (Systat Software Inc., San Jose, CA, USA). Val- by an independent safety monitoring board. Secondary endpoints ues were considered significant when p < 0¢05. include arrhythmias, biomarkers, MRI, and quality of life data. 3. Results 2.4. Cardiac magnetic resonance imaging data acquisition and analysis 3.1. Clinical-grade WJ-MSC production Magnetic resonance imaging (MRI) was performed in a state-of- the-art 1.5T clinical imaging system (InteraDB System; Philips, Eind- We obtained clinical grade WJ-MSCs with a 100% success rate hoven, NL) with the patient in the supine position, and a 16-element (n = 4), yielding a median of 6¢4 £ 108 WJ-MSCs, with 30 § 2 days phased-array coil placed over the chest. Images were acquired during from initial cord tissue explant culture to DP unit cryopreservation breath-holds with ECG gating. We used a segmented k-space steady- (Fig. 3A). In detail, MCB involved the isolation, selective expansion, 6 C. Prat-Vidal et al. / EBioMedicine 54 (2020) 102729 Fig. 3. Clinical-grade WJ-MSC characterisation. (a) Representative microscopic image of expanded WJ-MSCs culture. Scale bar = 100 mm. (b) Normal karyotype of a DP, correspond- ing to a representative cell line. (c) Immunophenotypic characteristics of four clinical-grade cell batches and average expression (mean § SEM). (d) Immunomodulation potential measured in four batches of clinical-grade WJ-MSCs, as the capacity to inhibit proliferation of polyclonal stimulated lymphocytes. (e) Microscopic images showing stimulated PBMC clumping, and clump reduction when co-cultured with WJ-MSCs. Arrowheads indicate clumps. Scale bars = 100 mm. PBMCs, peripheral blood mononuclear cells; WJ-MSCs, Whar- ton’s jelly mesenchymal stromal cells. and cryopreservation of WJ-MSCs. DP encompassed thawing a cryo- Table 2 preserved MCB unit, cell culture expansion, cryopreservation, and Specifications for preclinical construct and finished PeriCord human bioimplant. QCs. Various in-process controls, including microbiological testing, Preclinical PeriCord were performed throughout these steps (Table 1). After expansion, construct human bioimplant we evaluated sterility using European Pharmacopeia (Eur. Ph.), Myco- Active ingredient plasma, and endotoxin tests. In all cell batch productions, a cryopre- Viable cells Porcine MSCs Human WJ-MSCs served DP unit was generated for use as a control tube for the freeze- Density 0¢875 £ 106/cm2 (0¢62 0¢93) £ 106/cm2 thawing process, potency assay, adventitious virus testing, and kar- Dose 1¢75 £ 106 cells (7 15) £ 106 cells Viability 70% 70% yotyping. Observing no evidence of turbidity in any step of the three Excipient assayed runs in the Media Fill procedure, we considered the produc- Definition Vehicle Vehicle tion process aseptic. Hydrogel PuraMatrixTM Hydrogel PuraStatTM Table 2 Sucrose Sucrose DP viability was 97¢2 § 0¢5% (n = 4), with undetectable levels of Phosphate buffered saline PlasmalyteÒ Ò 148 Vehicle Decellularised, lyophilised, Decellularised, lyophilised, endotoxins and Mycoplasma DNA. No bacterial contamination or and irradiated human and irradiated human adventitious viruses were detected. To exclude any potential effects pericardium pericardium of the scale-up process on WJ-MSC genetic stability, we determined Vehicle size 2 cm2 12 16 cm2 G-banding karyotypes at early and late passages. At the cytogenetic Note: MSCs, mesenchymal stromal cells; WJ-MSCs, Wharton’s jelly mesenchymal level, all karyotypes showed unaltered patterns after accumulating stromal cells. 21 § 3 cumulative population doublings (Fig. 3B). Phenotypic profil- ing revealed a MSC-like pattern, with over 95% of cells strongly posi- tive for CD73, CD90, and CD105, and negative for CD31, CD45, and microscopic level, stimulated PBMCs clusters were reduced only HLA-DR (Fig. 3C). WJ-MSCs’ mesenchymal identity was conserved when WJ-MSCs were present, correlating to the immunopotency throughout expansion culture, with no differences observed between results obtained (Fig. 3E). initial and late passages. WJ-MSCs of different cell lines or passage numbers did not differ 3.2. Decellularised pericardial matrix processing in their ability to inhibit proliferation of polyclonal activated lympho- cytes. In all cases, clinical grade WJ-MSCs could inhibit proliferation The decellularisation protocol generated similar pale pericardial by 88¢7 § 6¢2% (range, 73¢8 99¢9% inhibition, n = 4; Fig. 3D). At the fragments, regardless of the initial sample size (Fig. 4A C, E, I, and K). C. Prat-Vidal et al. / EBioMedicine 54 (2020) 102729 7 Fig. 4. Pericardial tissue decellularisation. (a) Photograph showing native pericardium from deceased donor. (b) Native pericardium after dissection of superficial adhered adipose tissue. (c) Decellularised pericardial scaffolds after detergent treatment. Scale bars = 1 cm. (d and e) Masson’s trichrome staining of native (d) and decellularised (e) pericardium. Scale bars = 100 mm. (f and g) SEM-determined ultrastructure of native pericardium (f) and acellular pericardial matrix (g). Scale bars = 100 mm. (h k) Representative images of native pericardium (h and j) and decellularised pericardial scaffolds (i and k), showing immunostaining for col-III and elastin (both grey), and cell actin filaments with phalloidin (Phal, red). Nuclei were counterstained with DAPI (blue). Scale bars = 20 mm. (l) Genomic DNA quantification of native tissue, and 16 cm2 and 1 cm2 decellularised scaffolds, normal- ised to scaffold dry weight. *p < 0¢05 [paired Student’s t-test], native pericardium vs. decellularised scaffold. (m) Pore size measurements in decellularised pericardial scaffolds and vehicle. (n) Microscopic stiffness (Em) of pericardium measured with AFM in native tissues and decellularised scaffolds (n = 4). (o) Macroscopic stiffness (EM) of pericardium mea- sured at 20% stretch using tensile testing, for the same tissue conditions. Dashed lines in the graphs indicate the acceptance criteria for each parameter analysed. All data presented as mean § SEM. *p = 0¢046 [One-way ANOVA and Tukey’s post-hoc correction for multiple comparisons], between vehicle vs. decellularised pericardium (1 cm2). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Histological analysis with Masson’s trichrome staining showed no We evaluated the mechanical properties of pericardial tissue on two detectable cells and nuclei (Fig. 4D and E), and immunostaining scales: micromechanics using atomic force microscopy (AFM) (Fig. 4N), revealed no cytoskeletal elements or cellular nuclei (Fig. 4H K). and macromechanics by tensile testing (Fig. 4O). ECM stiffness did not Decellularised scaffolds maintained the intrinsic organisation and differ after decellularisation compared to native pericardium at the spatial three-dimensional distribution of the native matrix fibrils microscale or macroscale. Trehalose incubation, lyophilisation, and steri- (Fig. 4F and G), and the representative matrix proteins elastin, and lisation for obtaining the vehicle induced a slight decrease (~25%) in peri- type-III collagen were properly stained, indicating preservation of cardial scaffold stiffness at the microscale, and a smaller (~15%) non- extracellular matrix (ECM) protein components (Fig. 4I and K). Vali- significant softening at the macroscale (p = 0¢384 [one-way ANOVA]). dation of decellularisation was performed by processing four conse- Upon validation of the decellularisation protocol, we next vali- cutive pericardia, including samples of both ~16 cm2 and ~1 cm2. The dated the manufacture design of the final matrix (vehicle) by process- four decellularised batches met the proposed acceptance criteria for ing three consecutive pericardia. The three batches met the proposed all analysed parameters (Supplementary Table 1). DNA content was acceptance criteria for all analysed parameters in terms of serological <50 ng/mg of decellularised dry tissue, reaching a reduction of nearly and microbiological testing, sterility assessment, final quadrangular 100% in the final decellularised matrix (Fig. 4L). These minimal DNA size, and residual moisture. Cytotoxicity testing using decellularised, levels confirmed matrix suitability for clinical use, thus avoiding pos- lyophilised, and sterilised pericardial extracts revealed no cytotoxic sible adverse cell and host responses [25]. Matrix dimensions did not effects on cell viability (>70%, n = 3; p < 0¢001 [Student’s t-test]), significantly differ after decellularisation (2¢8 § 2¢9% global area demonstrating the suitability of the vehicle for clinical use. reduction; n = 5, p = 0¢209 [paired Student’s t-test]). The ~16-cm2 and ~1-cm2 decellularised scaffolds exhibited similar pore size 3.3. PeriCord cardiac bioimplant: scale-up and manufacturing process (26¢0 § 0¢6 mm vs. 27¢3 § 1¢2 mm; p = 0¢351 [paired Student’s t-test]) (Fig. 4M), and pore roundness ratio (0¢5 § 0¢006 vs. 0¢5 § 0¢01; PeriCord with clinically relevant dimensions (12 16 cm2) comprised p = 0¢230 [paired Student’s t-test]). Compared to decellularised scaf- human allogeneic WJ-MSCs colonising a human allogeneic decellularised folds, the vehicle exhibited smaller pore size (23¢1 § 1¢0 mm) and pericardium. Manufacturing process of PeriCord is schematically dis- bigger roundness ratio (0¢6 § 0¢01), but both parameters met the played in Fig. 1. Matrix colonisations were performed from post-thawed acceptance criteria demonstrating matrix equivalence. sucrose-conditioned cells (2¢6 £ 107 § 0¢1 £ 107 WJ-MSCs/mL; n = 37). 8 C. Prat-Vidal et al. / EBioMedicine 54 (2020) 102729 The initial thawed WJ-MSCs’ viability (96¢1 § 0¢1%) was unchanged fol- (n = 3). PeriCord RT stability period in its final container was defined lowing sucrose resuspension (92¢4 § 1¢0%), and remained within prod- as the maximum period for maintenance of WJ-MSCs viability at uct specifications even after 16 h at 2 8 °C (88¢8 § 1¢6%; n = 20). Cell 70% (product specification) and was set at 18 h (Fig. 5C). viability was verified after seeding in both FP (12 16 cm2) and small- To evaluate the impact of PeriCord transportation on the cell dose, scale replica (1 cm2), revealing no significant differences in WJ-MSCs’ FP and corresponding small-scale replica (n = 3 each) were manufac- viability when the same post-thawed and sucrose-conditioned cells tured and transported from BST to the Germans Trias i Pujol Univer- were seeded into three different matrix batches (FP: 79¢6 § 1¢2%; small- sity Hospital (12-km distance). After transportation, the transport scale replica: 78¢0 § 1¢1%; p = 0¢50 [paired Student’s t-test]) (Fig. 5A). solution (PlasmalyteÒÒ 148) was recovered, and the cells counted by Viable cells colonising matrix were also calculated, and no difference flow cytometry. The WJ-MSC dose defined in the batch release was between both sizes was observed (Fig. 5B). Moreover, cell viability did conserved by 98¢5% after transport (maximum variation, 4¢3%; aver- not change during sequential cell seeding on different matrix sectors age dose loss, 1¢5%) (Fig. 5D I). (n = 3 independent matrix batches; p = 0¢3 [Friedman test]). Following To validate the manufacture, three consecutive PeriCord batches the defined manufacturing protocol, WJ-MSCs’ viability in the FP was were processed, all of which met the acceptance criteria for cell dose 83¢1 § 0¢01% (n = 27). and viability. Sterility and Mycoplasma test were negative or unde- WJ-MSCs’ seeding efficiency and dose range in the FP were deter- tectable, and endotoxin levels were <4 EU/mL for all FPs. Environ- mined (n = 47) based on the cell retention capacity of pericardial mental microbial controls, pressure levels in qualified areas, and the matrices, analysed by calculating the difference between the initial number of particles fell within the operational range. No other rele- seeded WJ-MSCs and the total recovered cells after pH-balancing vant incidences were reported. washes detailed in the PeriCord manufacturing process. Average WJ- MSCs retention was >90%, corresponding to a density range of 3.4. PeriCord cardiac bioimplant: first-in-human patient implantation (0¢62 0¢93) £ 106 total viable WJ-MSCs/cm2, defining a dose in the range of (7 15) £ 106 total viable WJ-MSCs/FP. PeriCord was implanted in a 63-year-old male patient who suffered To validate PeriCord stability in the final packaging, we analysed an inferior myocardial infarct in 2004, which was conservatively WJ-MSC viability at different time-points, maintaining the FP at RT treated. He had a history of tobacco abuse, dyslipidaemia, and mild Fig. 5. PeriCord cardiac bioimplant. (a) Cell viability after colonisation was not significantly different between ~16-cm2 and ~1-cm2 matrices (n = 3 each). (b) Absolute number of via- ble colonising cells per cm2 was also similar in both matrix sizes. (c) Stability testing of PeriCord in final packaging at RT by determining WJ-MSCs’ viability using the LIVE/DEADTM method (n = 3). (d) Transport validation of three PeriCord batches and corresponding small-scale replicas. Colonisation percentage was determined by quantifying cells in recovered pH-balancing washes during manufacturing (pre-transportation), and in the PlasmalyteÒ 148 solution post-transportation. PeriCord (e) and small-replica (f) immobilised in the pri- mary packaging. (g and h) Secondary sterile packaging containing PlasmalyteÒ 148 solution. (i) A third container used for PeriCord transport. C. Prat-Vidal et al. / EBioMedicine 54 (2020) 102729 9 chronic obstructive pulmonary disease with sleep apnoea-hypopnoea end-diastolic volume was 159 mL and LV end-systolic volume 96 mL, syndrome. In March 2019, he presented progressive angina, and coro- resulting in reductions of 10% and 9%, respectively, relative to baseline nary angiography showed three-vessel disease, with an occluded right (Fig. 6E and F). coronary artery, unsuitable for revascularisation. Cardiac MRI showed transmural basal and mid inferior late gadolinium enhancement with 4. Discussion subendocardial extension towards the septum and lateral wall (scar mass = 7¢8 g, scar size scar mass normalised by total LV mass- = 5¢7%) Here we describe the complex translational process from preclini- (Fig. 6E). LV ejection fraction was 40%, with LV end-diastolic volume cal testing to first-in-human patient application of the PeriCord engi- 177 mL and LV end-systolic volume 106 mL. Having a clear indication neered 3D cardiac bioimplant, providing proof of principle that for revascularisation of the non-infarcted areas, the patient was offered PeriCord use in humans is feasible and preliminarily safe (Fig. 1). As PeriCord implantation in addition to the planned CABG. After receiving we adopt second-generation reparative therapies, such as cardiac tis- extensive information about the procedure, he gave written informed sue engineering, we must be careful to follow regulatory processes to consent. The first PeriCord manufactured for patient implantation con- ensure patient safety, and data validity and reproducibility. We will tained 12¢5 £ 106 viable WJ-MSCs in a 16-cm2 pericardial scaffold (via- next discuss the characteristics of the two components of the Peri- bility, 85¢4%; <0¢51 EU/mL) (Fig. 6A). Fig. 6B shows the inferior infarct Cord as compared with other approaches in the pipeline. during open-chest surgery, where the PeriCord was applied and First, we will comment on the cells, i.e., the active ingredient. Irre- secured with surgical glue at the four edges (Fig. 6C and D, and Supple- spective of origin and clinical purpose, cells used for tissue engineering mentary Video 1). The patient underwent CABG to the non-infarcted applications in human must comply with the Guidelines on Human Cell- area at left anterior descending, first diagonal, and oblique marginal. Based Medicinal Products (EMEA/CHMP/410869/2006) from the Euro- Early post-surgery follow-up was uneventful, except for moderate pean Medicines Agency, in terms of the cell population’s identity, respiratory distress and surgical suture intolerance unrelated to Peri- potency, purity, viability, safety, efficacy, and suitability for the intended Cord treatment. Post-surgery control echocardiogram showed no signs use. To date, autologous/allogenic hematopoietic stem cells (CD34+/ of pericarditis. No arrhythmic events were registered during the post- CD133+), and adult bone marrow/adipose tissue- or WJ-derived MSCs operative hospital stay. The patient had a one-week post-discharge are the only ATMPs listed on the European Medicines Agency website visit without significant findings. The PeriCord bioimplant does not (https://www.ema.europa.eu/en/human-regulatory/marketing-authori carry immunosuppression requirements due to low immunogenicity sation/advanced-therapies/advanced-therapy-classification/summaries- of the WJ-MSCs [26]. At the three-month follow-up, the patient was scientific-recommendations-classification-advanced-therapy-medicinal- alive and without hospital admissions. A three-month cardiac MRI products. Accessed July 30, 2019) intended for myocardial regeneration. revealed scar mass of 7¢1 g (scar size = 5¢3%), accounting for ~9% reduc- Of note, none of these are a combined material/cell product. Menasche  tion relative to baseline values. In terms of ventricular volumes, LV et al. pioneered the clinical translation of cardiac tissue engineering using Fig. 6. Surgical PeriCord implantation. (a) PeriCord batch for implantation, in primary packaging at the surgery theatre. (b) Non-revascularisable inferior infarct of the patient. (c and d) Expeditious PeriCord implantation secured by surgical glue droplets at the edges. (e and f) Representative short-axis delayed enhancement cardiac MRI obtained (e) before sur- gery and (f) at three-month follow-up. 10 C. Prat-Vidal et al. / EBioMedicine 54 (2020) 102729 embryonic stem cell (ESC)-derived cardiac progenitors embedded into a insufficient to determine whether a cardiac tissue engineered fibrin scaffold to treat patients with post-infarct scars [27,28]. However, approach requires full ex vivo cardiac myocyte and endothelial differ- ethical restrictions and the need for chronic immunosuppression may entiation, as has been attempted with cardiac-derived embryonic limit the clinical use of these cells. stem cells and induced pluripotent stem cells. This remains an open Clinical grade WJ-MSCs used in the PeriCord bioimplant could be question for future research. easily implemented elsewhere. WJ-MSCs exhibit a unique combina- In conclusion, in this first-in-human preliminary report, we tion of prenatal and postnatal cell properties, and thus constitute an describe the development and testing of a scalable, clinical-size, Peri- alternative to sources of stem cells with significant barriers of Cord cardiac bioimplant (Fig. 1). The PeriCord bioimplant has several immune rejection, and teratoma formation [29]. This cell type repre- advantages. It constitutes a unique combination of two human allo- sents an attractive candidate for a readily available off-the-shelf cel- geneic biological materials that can be stored off-the-shelf and used lular product for several reasons. WJ-MSCs spontaneously secrete upon request; does not require host immunosuppression; involves numerous pro-angiogenic factors with evidence of functional angio- minimal (if any) ethical issues; and incorporates WJ-MSCs, which genic potency in infarcted mammalian hearts [30]. WJ-MSCs are nat- exhibit karyotype stability, high immunomodulatory activity, lack of urally chemoattracted to cardiac tissue, and possess functional ability tumorigenesis, and substantial proliferative and differentiation to populate the ventricular myocardium [31]. In the mini-swine potential. Full safety confirmation will be achieved in the ongoing model, WJ-MSCs transplanted by a direct injection into the infarct PERISCOPE trial. differentiated into cardiomyocytes and endothelial cells [30]. In addi- tion, WJ-MSCs can be expanded ex vivo with high karyotype stability Declarations of Competing Interest [17], as shown here. Extensive research is being conducted with induced pluripotent stem cell-derived cardiomyocytes within engi- There are no conflicts of interest. neered heart tissue grafts. However, to our knowledge, translation to human application has not yet been accomplished [32,33]. Acknowledgements Secondly, we will discuss the decellularised pericardium, i.e., the vehicle. In tissue engineering, cells are embedded within a natural or The authors would like to acknowledge former and current mem- synthetic scaffold, avoiding the harsh infarcted milieu, while improv- bers of both Cell Therapy Service and Barcelona Tissue Bank (Banc de ing cell retention and survival compared to direct cell injection [12]. Sang i Teixits, Barcelona) for technical support, advice, and for experi- The scaffold biomaterial is crucial, and ideally should closely resemble mental help in responding to regulatory requests. The authors also the physiological cardiac ECM properties. Decellularised cardiac tissues thank research nurses for their valuable daily work. We specially provide a close match to the native, physiological microenvironment thank former and current members of the ICREC Research Lab for with minor changes in stiffness and preserving the composition and their unconditional support. We are very grateful to the donors who three-dimensional framework. Regarding mechanical properties, in a donated tissues altruistically, and to the patient who decided to par- head-to-head comparison of decellularised scaffolds from myocardial ticipate in the trial. and pericardial tissues (prepared following a technique similar to that presently described for the PeriCord), the decellularised pericardial Funding sources scaffold performed better [16]. showing preserved macromechanical and micromechanical pericardial scaffold properties upon the decellu- This work was supported by the La Marato  de TV3 Foundation larisation and recellularisation processes. Moreover, the pericardial [201502-30 and 201516-10]; Government of Catalonia: Agency for scaffold demonstrated better cell penetrance and retention, presum- Management of University and Research Grants (AGAUR) [2017-SGR- ably due to bigger pore size [16]. In the context of a preclinical MI 483], PERIS Program (Health Department) [SLT002/16/00234 and swine model, decellularised pericardial engineered grafts of human SLT002/16/00209] and CERCA Program; Catalan Society of Cardiol- origin integrated with the underlying myocardium, exhibiting signs of ogy; “La Caixa” Banking Foundation; grants from the Spanish Minis- neoinnervation and neovascularisation, and improved cardiac function try of Science, Innovation and Universities (MICINN) [SAF2017- post-MI [16]. Similar to WJ-MSCs, allogeneic decellularised pericardial 84324-C2-1-R, SAF2017-85574-R and DPI2017-83721-P]; Institute of matrices could be stored for off-the-shelf clinical use [34]. Health Carlos III (ISCIII) [PI18/00256 and PIC18/00014], Spanish Cell At present, the manufacturing of a PeriCord cardiac bioimplant by Therapy Network (TerCel) [RD16/0011/0006]; CIBER Cardiovascular repopulating a pericardial scaffold with WJ-MSCs is labour intensive [CB16/11/00403] projects, as a part of the National R&D&I Plan, and it and requires multiple steps with pre-specified QCs (Fig. 2). It can be was co-funded by ISCIII-Sub-Directorate General for Research Assess- achieved within eight working hours, producing a bioimplant valid ment and Promotion and the European Regional Development Fund for clinical use within the following 18 h. The same manufacturing (ERDF). The funders of the study had no role in study design, data col- process will be used for each patient enrolled in the phase I PERI- lection, data analysis, data interpretation, or writing of the report. SCOPE trial, which is currently in the patient enrolment stage. The reduction in scar mass observed at three months, although Author contributions remarkable, should be interpreted with caution and should not be used to construe a beneficial functional effect at this point. Indeed, ABG, CPV, PG, CGM, and SR conceived the initial study. CPV, LRG, this patient is a roll-in of the PERISCOPE trial, with a primary end- and MA designed and performed PeriCord scale-up, analysed, and point set on safety. Cardiac MRI data are part of the secondary end- interpreted experimental data. NNN, and CPV designed and per- points, and include both 3- and 12-month MRI. Nevertheless, cardiac formed decellularisation protocol, and histological experiments MRI accurately measures scarred and viable myocardium and repre- under the supervision of EA and AV. EA oversaw the decellularisation sents a useful tool for assessing dynamic changes [35]. Relative to the protocol logistics. IJ performed mechanical testing experiments and LV volume changes observed, our current understanding suggests analysed data under the supervision of RF and DN. PG, CGM, MMT, they likely result from the concomitant CABG than the PeriCord itself; and SR contributed to study design and data analysis. STZ, MIC, LR, nevertheless, future implants will shed more light in this issue. and CPV performed PeriCord production and quality controls under The presently reported PeriCord bioimplant included undifferen- the supervision of JV, LRG, and SQ. LRG supervised manufacturing tiated WJ-MSCs that, at the preclinical level, exhibited abilities to process and PeriCord batch release. PG, CGM, LRG, RC, and CPV pre- migrate, transdifferentiate, and induce favourable paracrine effects in pared documentation for both Regulatory Authority and Local Ethics underlying myocardium [29 31]. Current available data are Committee approvals. PG, and GC assisted in patient recruitment, C. 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