Mesenchymal stromal cells in the treatment of pediatric hematopoietic cell transplantation-related complications (graft vs. host disease, hemorrhagic cystitis, graft failure and poor graft function): a single center experience.

Objectives: To describe mesenchymal stromal cells (MSCs) in the treatment of hematopoietic stem cell transplantation (HSCT) complications and to assess its safety and efficacy. Methods: Single-center retrospective study (2016-2023). Patients under 20 years who received MSCs for the treatment of HSCT-related complications were included. Results: Thirty patients (53.7% boys), median age at transplant 11 years (range 2-19) were included. MSCs indications were: graft-vs.-host disease (GVHD) in 18 patients (60%), of them 13 had acute GVHD (43.3%) and 5 chronic GVHD (16.7%); Grade 3-4 hemorrhagic cystitis (HC) in 4 (13.3%); poor graft function (PGF) in 6 (20%), 5 of them receiving MSCs with a CD34 stem cell-boost coinfusion; graft failure (GF) in 2 (6.7%), to enhance engraftment after a subsequent HSCT. Infusion-related-adverse-events were not reported. Overall response (OR) was 83% (25/30); 44% of responders (11/25) showed complete response (CR). OR for GVHD, HC, PGF and GF was 83.3%, 100%, 66.7% and 100% respectively. Response rate was 40% (95% CI: 20-55) and 79% (95% CI: 57-89) at 15 and 30 days. With a median follow-up of 21 months (IQR11-52.5), overall survival (OS) was 86% (95% CI: 74-100) and 79% (95% CI: 65-95) at 6 and 12 months post-MSCs infusion. Conclusion: In our study, the most frequent indication of MSCs was refractory aGVHD (43.3%). Response rates were high (OR 83%) and safety profile was good.

Comparability exercise of critical quality attributes of clinical-grade human mesenchymal stromal cells from the Wharton's jelly: single-use stirred tank bioreactors versus planar culture systems.

BACKGROUND AIMS: The increasing demand of clinical-grade mesenchymal stromal cells (MSCs) for use in advanced therapy medicinal products (ATMPs) require a re-evaluation of manufacturing strategies, ensuring scalability from two-dimensional (2D) surfaces to volumetric (3D) productivities. Herein we describe the design and validation of a Good Manufacturing Practice-compliant 3D culture methodology using microcarriers and 3-L single-use stirred tank bioreactors (STRs) for the expansion of Wharton's jelly (WJ)-derived MSCs in accordance to current regulatory and quality requirements. METHODS: MSC,WJ were successfully expanded in 3D and final product characterization was in conformity with Critical Quality Attributes and product specifications previously established for 2D expansion conditions. RESULTS: After 6 days of culture, cell yields in the final product from the 3D cultures (mean 9.48 × 108 ± 1.07 × 107 cells) were slightly lower but comparable with those obtained from 2D surfaces (mean 9.73 × 108 ± 2.36 × 108 cells) after 8 days. In all analyzed batches, viability was >90%. Immunophenotype of MSC,WJ was highly positive for CD90 and CD73 markers and lacked of expression of CD31, CD45 and HLA-DR. Compared with 2D expansions, CD105 was detected at lower levels in 3D cultures due to the harvesting procedure from microcarriers involving trypsin at high concentration, and this had no impact on multipotency. Cells presented normal karyotype and strong immunomodulatory potential in vitro. Sterility, Mycoplasma, endotoxin and adventitious virus were negative in both batches produced. CONCLUSIONS: In summary, we demonstrated the establishment of a feasible and reproducible 3D bioprocess using single-use STR for clinical-grade MSC,WJ production and provide evidence supporting comparability of 3D versus 2D production strategies. This comparability exercise evaluates the direct implementation of using single-use STR for the scale-up production of MSC,WJ and, by extension, other cell types intended for allogeneic therapies.

Potency Assays: The 'Bugaboo' of Stem Cell Therapy.

Substantially manipulated cell-based products for human use are considered medicines and therefore regulatory authorities require extensive characterisation in terms of identity, purity and potency. The latter critical quality attribute is probably the most challenging to identify and measure, requiring provision that potency assays should reflect the intended mechanism of action and demonstrate the drugs' biological effect. However, in most cases, the mechanisms involved are not fully understood, making the definition and validation of suitable potency tests difficult, a 'bugaboo' quest to be feared. Although it is evident that much work is still needed in the scientific arena, the present chapter focuses on strategies currently used by developers of cell- and gene-based therapies to demonstrate potency of innovative medicines, the regulatory framework and need for standardisation seeking to demystify critical factors to consider when designing a potency assay.

Illustrative Potency Assay Examples from Approved Therapies.

Advanced therapy medicinal products (ATMP) encompass a new type of drugs resulting from the manipulation of genes, cells, and tissues to generate innovative medicinal entities with tailored pharmaceutical activity. Definition of suitable potency tests for product release are challenging in this context, in which the active ingredient is composed of living cells and the mechanism of action often is poorly understood. In this chapter, we present and discuss actual potency assays used for the release of representative commercial ATMP from each category of products (namely, KYMRIAH® (tisagenlecleucel), Holoclar® (limbal epithelial stem cells), and PROCHYMAL®/RYONCIL™ (remestemcel-L)). We also examine concerns related to the biological relevance of selected potency assays and challenges ahead for harmonization and broader implementation in compliance with current quality standards and regulatory guidelines.

Optimized reagents for immunopotency assays on mesenchymal stromal cells for clinical use.

Multipotent mesenchymal stromal cells (MSC) offer new therapeutic opportunities based on their ability to modulate an imbalanced 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).

Public Cord Blood Banks as a source of starting material for clinical grade HLA-homozygous induced pluripotent stem cells.

BACKGROUND: The increasing number of clinical trials for induced pluripotent stem cell (iPSC)-derived cell therapy products makes the production on clinical grade iPSC more and more relevant and necessary. Cord blood banks are an ideal source of young, HLA-typed and virus screened starting material to produce HLA-homozygous iPSC lines for wide immune-compatibility allogenic cell therapy approaches. The production of such clinical grade iPSC lines (haplolines) involves particular attention to all steps since donor informed consent, cell procurement and a GMP-compliant cell isolation process. METHODS: Homozygous cord blood units were identified and quality verified before recontacting donors for informed consent. CD34+ cells were purified from the mononuclear fraction isolated in a cell processor, by magnetic microbeads labelling and separation columns. RESULTS: We obtained a median recovery of 20.0% of the collected pre-freezing CD34+, with a final product median viability of 99.1% and median purity of 83.5% of the post-thawed purified CD34+ population. CONCLUSIONS: Here we describe our own experience, from unit selection and donor reconsenting, in generating a CD34+ cell product as a starting material to produce HLA-homozygous iPSC following a cost-effective and clinical grade-compliant procedure. These CD34+ cells are the basis for the Spanish bank of haplolines envisioned to serve as a source of cell products for clinical research and therapy.

Compliance in Non-Clinical Development of Cell-, Gene-, and Tissue-Based Medicines: Good Practice for Better Therapies.

The development of cell-, gene- and tissue engineering (CGT)-based therapies must adhere to strict pharmaceutical quality management standards, as for any other biological or small-molecule drug. However, early developments often failed to fully comply with good laboratory practices (GLP) in non-clinical safety studies. Despite an upward trend of positive opinions in marketing authorization applications, evidence of adherence to the principles of GLP is not openly reported; therefore, their relative impact on the overall quality of the product development program is unknown. Herein we investigated the actual degree of GLP implementation and the underlying factors impeding full compliance in non-clinical developments of CGT-based marketed medicines in the EU and USA, including (i) the co-existence of diverse quality management systems of more strategic value for small organizations, particularly current Good Manufacturing Practices n(GMP); (ii) lack of regulatory pressure to pursue GLP certification; and (iii) the involvement of public institutions lacking a pharmaceutical mindset and resources. As a final reflection, we propose conformity to good research practice criteria not as a doctrinaire impediment to scientific work, but as a facilitator of efficient clinical translation of more effective and safer innovative therapies.

Multicenter evaluation of the IL-3-pSTAT5 assay to assess the potency of cryopreserved stem cells from cord blood units: The BEST Collaborative study.

BACKGROUND: The IL-3-pSTAT5 assay, a new, rapid, and standardized flow-cytometry-based assay may compensate for several limitations of the colony-forming unit (CFU) assay typically used for stem cell potency assessments of cord blood units (CBU). We performed an inter-laboratory evaluation of the performance of this new assay. STUDY DESIGN AND METHODS: This Biomedical Excellence for Safer Transfusion (BEST) Collaborative multicenter, international study included 15 participants from public cord blood banks (CBBs), CBB-supporting research laboratories, and stem cell laboratories. To perform the IL-3-pSTAT5 assay, participating centers received reagents, instructions, and 10 blind CBU samples, including eight normal samples and two samples exposed to a transient warming event. We measured inter-laboratory agreement qualitatively (proportion of correctly classified samples) and quantitatively (coefficient of variation [CV], correlation coefficients, receiver operating characteristics (ROC) curve, and intraclass correlation coefficient [ICC]). RESULTS: The qualitative agreement was 97.3% (i.e., 107/110; Fleiss' kappa = 0.835). The average CV on a per-sample basis was 11.57% among all samples, 8.99% among normal samples, and on a per-center basis was 9.42% among normal samples. In a correlation matrix that compared results across centers, the mean Pearson's correlation coefficient was 0.88 (standard deviation = 0.04). The ICC was 0.83 (95% confidence interval = 0.68-0.95). The area under the curve (AUC) from the ROC curve was 0.9974. DISCUSSION: Excellent qualitative and quantitative agreement was exhibited across laboratories. The IL-3-pSTAT5 assay may therefore be implemented in flow cytometry laboratories to rapidly and reliably provide standardized measures of stem cell potency in CBUs.

Cryopreservation of unrelated donor hematopoietic stem cells: the right answer for transplantations during the COVID-19 pandemic?

Cryopreservation was recommended to ensure continuity of unrelated donor (UD) hematopoietic stem cell transplantation (HSCT) during COVID-19 pandemic. However, its impact on clinical outcomes and feasibility was not well known. We compared 32 patients who underwent UD HSCT using cryopreserved peripheral blood stem cells (PBSC) during the COVID-19 pandemic with 32 patients who underwent UD HSCT using fresh PBSC in the previous period. Median neutrophil engraftment was 17.5 and 17.0 days with cryopreserved and fresh grafts, respectively. Non-significant delays were found in platelet recovery days (25.5 versus 19.0; P = 0.192) and full donor chimerism days (35.0 and 31.5; P = 0.872) using cryopreserved PBSC. The rate of acute graft-versus-host disease at 100 days was 41% (95% CI [21-55%]) in cryopreserved group versus 31% (95% CI [13-46%]) in fresh group (P = 0.380). One-hundred days progression-relapse free survival and overall survival did not differ significantly. During COVID-19 pandemic, six frozen UD donations were not transfused and logistical and clinical issues regarding cryopreservation procedure, packaging, and transporting appeared. In summary, UD HSCT with cryopreserved PBSC was safe during this challenging time. More efforts are needed to ensure that all frozen grafts are transplanted and cryopreservation requirements are harmonized.

Diagnostic performance of the ClearLLab 10C B cell tube.

INTRODUCTION: Pre-analytical and analytical errors can threaten the reliability of flow cytometry (FC) results. A potential solution to some of these is the use of dry, pre-mixed antibodies, such as the ClearLLab 10C system. The purpose of the present study was to compare the diagnostic performance of the ClearLLab 10C B cell tube with that of our standard laboratory practice. METHODS: We compared the diagnoses made with the ClearLLab 10C B cell tube (experimental strategy) with those made with standard laboratory practice (standard strategy). Samples were selected aiming for representation of the full spectrum of B cell disorders, with an emphasis on mature B cell malignancies, as well as healthy controls. RESULTS: We included 116 samples (34 normal controls, 4 acute lymphoblastic leukemias, 54 mature lymphoproliferative disorders in peripheral blood and bone marrow, 3 myelomas, 6 bone marrow samples with involvement by lymphoma and 1 with elevated hematogone count, 14 lymph node samples, 1 cerebrospinal fluid, and 1 pleural effusion). There were two diagnostic errors (1.7%). The agreement between the two strategies in the percentage of CD19 cells and fluorescence intensity of CD5, CD19, CD20, CD200, and CD10 was very good. CONCLUSIONS: In this study, the ClearLLab 10C B cell tube performed similarly to our standard laboratory practice to diagnose and classify mature B cell malignancies.

Design and validation of a consistent and reproducible manufacture process for the production of clinical-grade bone marrow-derived multipotent mesenchymal stromal cells.

BACKGROUND: Multipotent mesenchymal stromal cells (MSC) have achieved a notable prominence in the field of regenerative medicine, despite the lack of common standards in the production processes and suitable quality controls compatible with Good Manufacturing Practice (GMP). Herein we describe the design of a bioprocess for bone marrow (BM)-derived MSC isolation and expansion, its validation and production of 48 consecutive batches for clinical use. METHODS: BM samples were collected from the iliac crest of patients for autologous therapy. Manufacturing procedures included: (i) isolation of nucleated cells (NC) by automated density-gradient centrifugation and plating; (ii) trypsinization and expansion of secondary cultures; and (iii) harvest and formulation of a suspension containing 40 ± 10 × 10(6) viable cells. Quality controls were defined as: (i) cell count and viability assessment; (ii) immunophenotype; and (iii) sterility tests, Mycoplasma detection, endotoxin test and Gram staining. RESULTS: A 3-week manufacturing bioprocess was first designed and then validated in 3 consecutive mock productions, prior to producing 48 batches of BM-MSC for clinical use. Validation included the assessment of MSC identity and genetic stability. Regarding production, 139.0 ± 17.8 mL of BM containing 2.53 ± 0.92 × 10(9) viable NC were used as starting material, yielding 38.8 ± 5.3 × 10(6) viable cells in the final product. Surface antigen expression was consistent with the expected phenotype for MSC, displaying high levels of CD73, CD90 and CD105, lack of expression of CD31 and CD45 and low levels of HLA-DR. Tests for sterility, Mycoplasma, Gram staining and endotoxin had negative results in all cases. DISCUSSION: Herein we demonstrated the establishment of a feasible, consistent and reproducible bioprocess for the production of safe BM-derived MSC for clinical use.