Compliance with Good Manufacturing Practice in the Assessment of Immunomodulation Potential of Clinical Grade Multipotent Mesenchymal Stromal Cells Derived from Wharton’s Jelly

Cell culture; Cellular therapy; Good manufacturing practiceCultiu cel·lular; Teràpia cel·lular; Bones pràctiques de manufacturaCultivo celular; Terapia celular; Buenas prácticas de fabricaciónThe selection of assays suitable for testing the potency of clinical grade multipotent mesenchymal stromal cell (MSC)-based products and its interpretation is a challenge for both developers and regulators. Here, we present a bioprocess design for the production of Wharton's jelly (WJ)-derived MSCs and a validated immunopotency assay approved by the competent regulatory authority for batch release together with the study of failure modes in the bioprocess with potential impact on critical quality attributes (CQA) of the final product. Methods: The lymphocyte proliferation assay was used for determining the immunopotency of WJ-MSCs and validated under good manufacturing practices (GMP). Moreover, failure mode effects analysis (FMEA) was used to identify and quantify the potential impact of different unexpected situations on the CQA. Results: A production process based on a two-tiered cell banking strategy resulted in batches with sufficient numbers of cells for clinical use in compliance with approved specifications including MSC identity (expressing CD73, CD90, CD105, but not CD31, CD45, or HLA-DR). Remarkably, all batches showed high capacity to inhibit the proliferation of activated lymphocytes. Moreover, implementation of risk management tools led to an in-depth understanding of the manufacturing process as well as the identification of weak points to be reinforced. Conclusions: The bioprocess design showed here together with detailed risk management and the use of a robust method for immunomodulation potency testing allowed for the robust production of clinical-grade WJ-MSCs under pharmaceutical standards

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

Immunomodulation; Mesenchymal stromal cells; Quality & regulatory complianceImmunomodulació; Cèl·lules estromals mesenquimàtiques; Qualitat i compliment normatiuInmunomodulación; Células estromales mesenquimales; Calidad y cumplimiento normativoMultipotent 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.Open Access Funding provided by Universitat Autonoma de Barcelona. This work has been developed in the context of Red Española de Terapias Avanzadas (TERAV, expedient no. RD21/0017/0022) funded by Instituto de Salud Carlos III (ISCIII) in the context of NextGenerationEU’s Recovery, Transformation and Resilience Plan and by the Commission for Universities and Research of the Department of Innovation, Universities, and Enterprise of the Generalitat de Catalunya (2017 SGR 719)

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

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

Reparació prenatal del mielomeningocele mitjançant cèl·lules mesenquimals estromals de líquid amniòtic en model oví

Aquest treball de tesi doctoral s’emmarca en un camp de recerca dins la medicina regenerativa, l'objectiu del qual és la reparació prenatal del Mielomeningocele (MMC), que és la forma més comú i severa d’espina bífida. Aquesta malaltia congènita es caracteritza per un defecte en el tancament de la part posterior del tub neural durant el període embrionari, quedant la medul·la espinal i les seves arrels nervioses exposades al medi uterí. En conseqüència, el teixit neural pateix un deteriorament anatòmic i funcional progressiu sever, acompanyat d’una pèrdua de líquid cefaloraquidi per la zona del defecte, que dóna lloc al desenvolupament de la malformació de Chiari II i hidrocefàlia. Les tècniques utilitzades actualment en clínica han permès dur a terme cirurgies fetals amb l’objectiu de cobrir o reparar el defecte. D’aquesta manera s’aconsegueix frenar el deteriorament del teixit neural, revertir la malformació de Chiari i reduir la necessitat de derivacions ventriculo-peritoneals per tractar l’hidrocefàlia. Tot i la millora en l’estat neurocognitiu, motor i urinari, es requereixen cirurgies correctores per l’aparició de deformitats esquelètiques sobretot durant la infantesa. La manca d’estructures òssies a la part posterior de les vertebres on fixar els sistemes metàl·lics d’estabilització espinal, dificulta enormement aquestes cirurgies. En la tesi que aquí es presenta s’ha treballat en el desenvolupament d’un producte d’enginyeria tissular, basat en cèl·lules mesenquimals estromals autòlogues aïllades de líquid amniòtic (AF-oMSCs), en combinació amb diferents tipus de matrius biocompatibles en un model experimental oví de MMC induït quirúrgicament. El producte cel·lular s’ha generat a partir de mostres de líquid amniòtic, obtingudes en el moment de la cirurgia d’inducció del MMC, que s’han cultivat durant les aproximadament tres setmanes que la separen de la cirurgia de reparació. S’han caracteritzat els paràmetres cinètics, fenotípics i funcionals de les AF-oMSCs aïllades i expandides ex vivo. Així mateix, s’han posat al punt metodologies pel marcatge i seguiment cel·lular utilitzant micropartícules d’òxid de ferro (MPIOs) i la transducció amb partícules virals que codifiquen per la proteïna verda fluorescent (eGFP). S’han avaluat tres tipus de biomatrius compostes d’àcid polilàctic-co-glicòlic (PLGA), fibrina i matriu òssia desmineralitzada (DBM). Els resultats d’aquest projecte demostren la possibilitat de generar, a partir dels constructes compostos per una barreja d’AF-oMSCs, DBM i Fibrina, una estructura òssia similar a un arc vertebral posterior amb moll d’os a l’interior, ben integrat anatòmicament i rodejat per teixit connectiu, teixit adipós i recobert per pell.The PhD project presented here aims at offering a new srategy for the repair of Myelomeningocele (MMC) using tools from Regenerative Medicine and Tissue Engineering. MMC (or spina bifida) is a congenital condition characterised by a defective closure of the neural tube during embryonic development that results in a malformation of the spinal cord, which is exposed to the uterine environment. As a consequence, the neural tissue suffers anatomic and functional degeneration along with loss of cephaloraquideum liquid from the defect site, resulting in Chiari II malformation and hydrocephalus. Approaches currently used clinically have enabled the successful performance of fetal surgery with the objective of covering and repair the defect up to some extent. This has permitted to block the progression of degenerative processes in the neural tissue, reverting Chiari malformations, and reduce the need of ventricular-peritoneal derivations for treating hydrocephalus. Despite the improvements achieved to date (particularly with respect to neurocognitive, motor and urinary status), additional corrective interventions are still required for the treatment of skeletal malformations during infancy. The lack of bony structures at the posterior side of vertebrae for fixing metallic stabilisers largely difficult such approaches. The work presented here addresses the development of tissue engineering product based on the use of Mesenchymal Stromal Cells isolated from the amniotic fluid (AF-oMSCs) combined with different types of biocompatible extracellular matrices and tested in an ovine animal model of experimental MMC induced surgically. The cellular component of the tissue engineering product investigated in this work was derived from samples of amniotic fluid harvested at the very moment of the MMC-inducing surgery, then expanded in vitro for a three-week long period, and finally administered to the animals for the treatment of MMC. A comprehensive characterisation of ex vivo expanded AF-oMSCs was performed, including phenotypic profile, multipotentiality, and the analysis of kinetic parameters of cell cultures. Furthermore labelling methods of AF-oMSCs for in vivo cell fate tracking were assessed in sheep, including Magnetic Particles of Iron Oxide (MPIO) and transduction with viruses encoding the enhanced Green Fluorescent Protein (eGFP). Regarding the scaffolds, polylactic-co-glycolic acid (PLGA), fibrin and demineralised bone matrix (DBM) were assayed in vitro and tested in vivo. The feasibility of generating a posterior vertebral arch-like structure in vivo after treatment of MMC with a construct composed of AF-oMSCs, DBM, and fibrin was demonstrated. Moreover, proper anatomic integration with adjacent connective and adipose tissues, recovered with skin, resembling native tissue was observed

Reparació prenatal del mielomeningocele mitjançant cèl·lules mesenquimals estromals de líquid amniòtic en model oví

Aquest treball de tesi doctoral s'emmarca en un camp de recerca dins la medicina regenerativa, l'objectiu del qual és la reparació prenatal del Mielomeningocele (12C), que és la forma més comú i severa d'espina bífida. Aquesta malaltia congènita es caracteritza per un defecte en el tancament de la part posterior del tub neural durant el període embrionari, quedant la medul·la espinal i les seves arrels nervioses exposades al medi uterí. En conseqüència, el teixit neural pateix un deteriorament anatòmic i funcional progressiu sever, acompanyat d'una pèrdua de líquid cefaloraquidi per la zona del defecte, que dóna lloc al desenvolupament de la malformació de Chiari II i hidrocefàlia. Les tècniques utilitzades actualment en clínica han permès dur a terme cirurgies fetals amb l'objectiu de cobrir o reparar el defecte. D'aquesta manera s'aconsegueix frenar el deteriorament del teixit neural, revertir la malformació de Chiari i reduir la necessitat de derivacions ventriculo-peritoneals per tractar l'hidrocefàlia. Tot i la millora en l'estat neurocognitiu, motor i urinari, es requereixen cirurgies correctores per l'aparició de deformitats esquelètiques sobretot durant la infantesa. La manca d'estructures òssies a la part posterior de les vertebres on fixar els sistemes metàl·lics d'estabilització espinal, dificulta enormement aquestes cirurgies. En la tesi que aquí es presenta s'ha treballat en el desenvolupament d'un producte d'enginyeria tissular, basat en cèl·lules mesenquimals estromals autòlogues aïllades de líquid amniòtic (AF-oMSCs), en combinació amb diferents tipus de matrius biocompatibles en un model experimental oví de 12C induït quirúrgicament. El producte cel·lular s'ha generat a partir de mostres de líquid amniòtic, obtingudes en el moment de la cirurgia d'inducció del 12C, que s'han cultivat durant les aproximadament tres setmanes que la separen de la cirurgia de reparació. S'han caracteritzat els paràmetres cinètics, fenotípics i funcionals de les AF-oMSCs aïllades i expandides ex vivo. Així mateix, s'han posat al punt metodologies pel marcatge i seguiment cel·lular utilitzant micropartícules d'òxid de ferro (MPIOs) i la transducció amb partícules virals que codifiquen per la proteïna verda fluorescent (eGFP). S'han avaluat tres tipus de biomatrius compostes d'àcid polilàctic-co-glicòlic (PLGA), fibrina i matriu òssia desmineralitzada (DBM). Els resultats d'aquest projecte demostren la possibilitat de generar, a partir dels constructes compostos per una barreja d'AF-oMSCs, DBM i Fibrina, una estructura òssia similar a un arc vertebral posterior amb moll d'os a l'interior, ben integrat anatòmicament i rodejat per teixit connectiu, teixit adipós i recobert per pellThe PhD project presented here aims at offering a new srategy for the repair of Myelomeningocele (MMC) using tools from Regenerative Medicine and Tissue Engineering. MMC (or spina bifida) is a congenital condition characterised by a defective closure of the neural tube during embryonic development that results in a malformation of the spinal cord, which is exposed to the uterine environment. As a consequence, the neural tissue suffers anatomic and functional degeneration along with loss of cephaloraquideum liquid from the defect site, resulting in Chiari II malformation and hydrocephalus. Approaches currently used clinically have enabled the successful performance of fetal surgery with the objective of covering and repair the defect up to some extent. This has permitted to block the progression of degenerative processes in the neural tissue, reverting Chiari malformations, and reduce the need of ventricular-peritoneal derivations for treating hydrocephalus. Despite the improvements achieved to date (particularly with respect to neurocognitive, motor and urinary status), additional corrective interventions are still required for the treatment of skeletal malformations during infancy. The lack of bony structures at the posterior side of vertebrae for fixing metallic stabilisers largely difficult such approaches. The work presented here addresses the development of tissue engineering product based on the use of Mesenchymal Stromal Cells isolated from the amniotic fluid (AF-oMSCs) combined with different types of biocompatible extracellular matrices and tested in an ovine animal model of experimental MMC induced surgically. The cellular component of the tissue engineering product investigated in this work was derived from samples of amniotic fluid harvested at the very moment of the MMC-inducing surgery, then expanded in vitro for a three-week long period, and finally administered to the animals for the treatment of MMC. A comprehensive characterisation of ex vivo expanded AF-oMSCs was performed, including phenotypic profile, multipotentiality, and the analysis of kinetic parameters of cell cultures. Furthermore labelling methods of AF-oMSCs for in vivo cell fate tracking were assessed in sheep, including Magnetic Particles of Iron Oxide (MPIO) and transduction with viruses encoding the enhanced Green Fluorescent Protein (eGFP). Regarding the scaffolds, polylactic-co-glycolic acid (PLGA), fibrin and demineralised bone matrix (DBM) were assayed in vitro and tested in vivo. The feasibility of generating a posterior vertebral arch-like structure in vivo after treatment of MMC with a construct composed of AF-oMSCs, DBM, and fibrin was demonstrated. Moreover, proper anatomic integration with adjacent connective and adipose tissues, recovered with skin, resembling native tissue was observed

Compliance with Good Manufacturing Practice in the Assessment of Immunomodulation Potential of Clinical Grade Multipotent Mesenchymal Stromal Cells Derived from Wharton’s Jelly

Background: The selection of assays suitable for testing the potency of clinical grade multipotent mesenchymal stromal cell (MSC)-based products and its interpretation is a challenge for both developers and regulators. Here, we present a bioprocess design for the production of Wharton’s jelly (WJ)-derived MSCs and a validated immunopotency assay approved by the competent regulatory authority for batch release together with the study of failure modes in the bioprocess with potential impact on critical quality attributes (CQA) of the final product. Methods: The lymphocyte proliferation assay was used for determining the immunopotency of WJ-MSCs and validated under good manufacturing practices (GMP). Moreover, failure mode effects analysis (FMEA) was used to identify and quantify the potential impact of different unexpected situations on the CQA. Results: A production process based on a two-tiered cell banking strategy resulted in batches with sufficient numbers of cells for clinical use in compliance with approved specifications including MSC identity (expressing CD73, CD90, CD105, but not CD31, CD45, or HLA-DR). Remarkably, all batches showed high capacity to inhibit the proliferation of activated lymphocytes. Moreover, implementation of risk management tools led to an in-depth understanding of the manufacturing process as well as the identification of weak points to be reinforced. Conclusions: The bioprocess design showed here together with detailed risk management and the use of a robust method for immunomodulation potency testing allowed for the robust production of clinical-grade WJ-MSCs under pharmaceutical standards

Compliance with good manufacturing practice in the assessment of immunomodulation potential of clinical grade multipotent mesenchymal stromal Cells Derived from Wharton's Jelly

The selection of assays suitable for testing the potency of clinical grade multipotent mesenchymal stromal cell (MSC)-based products and its interpretation is a challenge for both developers and regulators. Here, we present a bioprocess design for the production of Wharton's jelly (WJ)-derived MSCs and a validated immunopotency assay approved by the competent regulatory authority for batch release together with the study of failure modes in the bioprocess with potential impact on critical quality attributes (CQA) of the final product. Methods: The lymphocyte proliferation assay was used for determining the immunopotency of WJ-MSCs and validated under good manufacturing practices (GMP). Moreover, failure mode effects analysis (FMEA) was used to identify and quantify the potential impact of different unexpected situations on the CQA. Results: A production process based on a two-tiered cell banking strategy resulted in batches with sufficient numbers of cells for clinical use in compliance with approved specifications including MSC identity (expressing CD73, CD90, CD105, but not CD31, CD45, or HLA-DR). Remarkably, all batches showed high capacity to inhibit the proliferation of activated lymphocytes. Moreover, implementation of risk management tools led to an in-depth understanding of the manufacturing process as well as the identification of weak points to be reinforced. Conclusions: The bioprocess design showed here together with detailed risk management and the use of a robust method for immunomodulation potency testing allowed for the robust production of clinical-grade WJ-MSCs under pharmaceutical standards