Cell-based Medicinal Products Research Articles

Safety of a Medicinal Product Based on Human Glial Progenitor Cells: A Pilot Study of Retrobulbar Administration in C57BL/6J Mice

INTRODUCTION. Stem cell therapy is a promising treatment method for various diseases and injuries, but its safety has yet to be determined. Therefore, studying the safety of administering a xenogeneic cell-based medicinal product (CBMP) into the retro-orbital venous sinus is essential for developing protocols for further studies of potential medicinal products for neurological conditions.AIM. The aim of the study was to determine the optimal dose of a CBMP derived from glial progenitor cells (GPCs) and to evaluate its safety during retrobulbar administration in C57BL/6J mice.MATERIALS AND METHODS. GPCs were derived from human induced pluripotent stem cells by stepwise differentiation and cultured in DMEM/F12 supplemented with epidermal growth factor and ciliary neurotrophic factor. Matrigel was used as a substrate. GPCs were injected into the retro-orbital venous sinus of male C57BL/6J mice under isoflurane anaesthesia once a week for two months. The study analysed changes in biochemical blood parameters and behaviour. The quantities of activated astrocytes and glial cells were determined by postmortem immunohistochemical staining.RESULTS. The administration of GPCs at a dose of 500×103 cells/mouse, which was selected using literature data, induced an increase in the plasma levels of ala nine aminotransferase and aspartate aminotransferase. This could indicate cell damage and the development of inflammatory reactions. At doses reduced to one-third the initial GPC concentration or lower, the biochemical blood parameters of the treatment groups did not differ significantly from those of the control group. There were no significant differences in neuroinflammatory markers between the groups receiving GPCs at different doses, except for an increase in astrocyte activation at a dose of 150×103 cells/mouse, which could potentially indicate inflammatory processes in the brain. The study detected no pathological changes in the brain or cell damage markers in the blood of mice after retrobulbar GPC injections of 15×103 or 50×103 cells/mouse.CONCLUSIONS. The study results indicate that long-term therapy with GPCs is potentially safe for mice if the dose is optimal. The authors suggest using the optimal doses and the administration route established in this study for further research into the safety of intravenous administration of CBMPs for neurological conditions.

Cell-based medicinal products: a review of current research

INTRODUCTION. Cell therapies and tissue-engineered products are aimed at patients with severe conditions (genetic and neurodegenerative disorders, cancers, musculoskeletal injuries, burns, etc.) that lack alternative treatment options. Analysis of clinical efficacy data on cellbased medicinal products is important for understanding their translational potential in personalised medicine.AIM. This study aimed to review key trends in cell therapy, analyse data on approved cell therapies and tissue-engineered products, and assess challenges and prospects for their use.DISCUSSION. This article analyses data on the composition of cell therapies and tissue-engineered products, indications for their use, and the results of clinical studies. Cell-based medicinal products are derived from autologous or allogeneic mesenchymal and limbal stem cells, epithelial cells, chondrocytes, native or genetically engineered haematopoietic stem cells, genetically engineered lymphocytes (CAR-T, CAR-NK), etc. Medicinal products based on cell technologies have been approved in many countries, including the USA (approximately 30), the European Union (approximately 20), Japan (18), South Korea (15), etc. As of today, two cell therapies have been granted marketing authorisation in the Russian Federation. The first is based on CAR-T cells (a gene therapy product), and the other is based on chondrocytes (a cell-based medicinal product); the latter has been developed in Russia. The main advantages of cell therapy products include higher efficacy and fewer adverse drug reactions in comparison with standard treatment modalities. The main challenges of cell therapy include the risks of immune reactions and mutagenesis associated with lentiviral vectors or CRISPR/Cas9 technology, as well as limited efficacy of CAR-T and CAR-NK cells due to immunosuppressive properties of tumour microenvironment.CONCLUSIONS. In comparison with conventional treatment approaches, the use of cell therapies and tissue-engineered products can help effectively eliminate defects in various body tissues, avoid highly invasive surgical interventions, and reduce regeneration time. Thus, ensuring development of similar but at the same time more affordable Russian medicinal products can bring great benefits for the healthcare system of the Russian Federation.

Experience in the production and clinical application of the cell-based medicinal product Easytense® for the repair of cartilage defects of the human knee

INTRODUCTION. The current cell-based cartilage repair methods, such as autologous chondrocyte transplantation, are not sufficiently effective, and the surgery is painful and traumatic. Therefore, there is a need for a more effective cell therapy product with a minimally invasive surgical procedure for its implantation into the patient.AIM. This study aimed to develop a manufacturing technology for the production of an autologous cell-based medicinal product (CBMP) comprising three-dimensional structures (3D-spheroids) based on chondrocytes isolated from the patient’s cartilage tissue, as well as to evaluate its clinical efficacy.MATERIALS AND METHODS. Autologous chondrocytes isolated from the patient’s cartilage biopsy were propagated in monolayer culture to obtain the required number of cells. Subsequently, the chondrocytes were cultivated on plates with a non-adhesive coating to form 3D spheroids. All CBMP production steps were performed under aseptic conditions in cell culture isolators. The authors used phase-contrast microscopy and immunohistochemical staining with specific fluorescence-labelled antibodies to characterise chondrocyte phenotypes at different stages of cultivation. Genetic stability was controlled by karyotyping. The efficacy of Easytense® was evaluated in a clinical trial using specialised functional tests and the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score. The primary efficacy endpoint was a change in the overall score on the Knee Injury and Osteoarthritis Outcome Score (KOOS).RESULTS. A manufacturing technology without using animal sera, growth factors, cytokines, or other additives was developed for the production of the autologous CBMP Easytense®. Karyological data confirmed that the chondrocytes retained genetic stability for 3 passages in monolayer culture. When cultured as 3D spheroids, the chondrocytes produced cartilage extracellular matrix proteins (type II collagen, aggrecan), thus acquiring the ability to repair damaged cartilage. The clinical trial demonstrated a statistically significant improvement in knee cartilage 12 months after the transplantation of 3D spheroids derived from autologous chondrocytes. The mean change in the overall KOOS score was 23.8±15.9.CONCLUSIONS. The clinical trial results indicate that Easytense® is highly effective for cartilage repair. Based on these results, the CBMP has been granted marketing authorisation and introduced into clinical practice in the Russian Federation. Easytense® has the potential to replace endoprosthetics and expensive surgeries abroad.

Stain-Free Approach to Determine and Monitor Cell Heath Using Supervised and Unsupervised Image-Based Deep Learning

Cell-based medicinal products (CBMPs) are a growing class of therapeutics that promise new treatments for complex and rare diseases. Given the inherent complexity of the whole human cells comprising CBMPs, there is a need for robust and fast analytical methods for characterization, process monitoring, and quality control (QC) testing during their manufacture. Existing techniques to evaluate and monitor cell quality typically constitute labor-intensive, expensive, and highly specific staining assays. In this work, we combine image-based deep learning with flow imaging microscopy (FIM) to predict cell health metrics using cellular morphology “fingerprints” extracted from images of unstained Jurkat cells (immortalized human T-lymphocyte cells). A supervised (i.e., algorithm trained with human-generated labels for images) fingerprinting algorithm, trained on images of unstained healthy and dead cells, provides a robust stain-free, non-invasive, and non-destructive method for determining cell viability. Results from the stain-free method are in good agreement with traditional stain-based cytometric viability measurements. Additionally, when trained with images of healthy cells, dead cells and cells undergoing chemically induced apoptosis, the supervised fingerprinting algorithm is able to distinguish between the three cell states, and the results are independent of specific treatments or signaling pathways. We then show that an unsupervised variational autoencoder (VAE) algorithm trained on the same images, but without human-generated labels, is able to distinguish between samples of healthy, dead and apoptotic cells along with cellular debris based on learned morphological features and without human input. With this, we demonstrate that VAEs are a powerful exploratory technique that can be used as a process monitoring analytical tool.

Lessons learned from contamination with endotoxin originated from the supplement in the cell culture medium

IntroductionEndotoxin is a typical pyrogen derived from the outer membrane of Gram-negative bacteria. In fabricating cell-based medicinal products, it is necessary to control endotoxin in the process and the products. In the quality control tests of our clinical study, endotoxin concentration in the culture supernatant of autologous oral mucosal epithelial cell sheets exceeded the criterion value. Therefore, endotoxin measurements were conducted to clarify the cause of the endotoxin contamination. MethodsThe reagents used to prepare the culture medium, the unused culture medium, and the culture supernatants were diluted with pure water. Endotoxin concentrations in the diluted samples were measured. ResultsEndotoxin was detected in both the unused culture medium and the culture supernatant of the epithelial cell sheets at higher concentrations than the criterion value. Therefore, endotoxin concentrations in the reagents used to prepare the culture medium were measured and were found to be below the criterion value, except for cholera toxin. On the other hand, three lots of cholera toxin products were used for the measurement, and the endotoxin concentrations were higher than the criterion value. The results indicate that the endotoxin contamination is caused by the cholera toxin product. ConclusionsTo prevent endotoxin contamination in cell-based medicinal products, endotoxin concentrations in reagents used for the fabrication should be measured in the facility conducting clinical research or confirmed by an adequate certificate of analysis from the manufacturers of the reagents.

Potency assay to predict the anti-inflammatory capacity of a cell therapy product for macrophage-driven diseases: overcoming the challenges of assay development and validation

BackgroundGiven the high level of product complexity and limited regulatory guidance, designing and implementing appropriate potency assays is often the most challenging part of establishing a quality control testing matrix for a cell-based medicinal product. Among the most elusive tasks are the selection of suitable read-out parameters, the development of assay designs that most closely model the pathophysiological conditions, and the validation of the methods. Here we describe these challenges and how they were addressed in developing an assay that measures the anti-inflammatory potency of mesenchymal stromal cells (MSCs) in an M1 macrophage-dominated inflammatory environment. MethodsAn in vitro inflammation model was established by coculturing skin-derived ABCB5+ MSCs with THP-1 monocyte-derived M1-polarized macrophages. Readout was the amount of interleukin 1 receptor antagonist (IL-1RA) secreted by the MSCs in the coculture, measured by an enzyme-linked immunosorbent assay. ResultsIL-1RA was quantified with guideline-concordant selectivity, accuracy and precision over a relevant concentration range. Consistent induction of the macrophage markers CD36 and CD80 indicated successful macrophage differentiation and M1 polarization of THP-1 cells, which was functionally confirmed by release of proinflammatory tumor necrosis factor α. Testing a wide range of MSC/macrophage ratios revealed the optimal ratio for near-maximal stimulation of MSCs to secrete IL-1RA, providing absolute maximum levels per individual MSC that can be used for future comparison with clinical efficacy. Batch release testing of 71 consecutively manufactured MSC batches showed a low overall failure rate and a high comparability between donors. ConclusionsWe describe the systematic development and validation of a therapeutically relevant, straightforward, robust and reproducible potency assay to measure the immunomodulatory capacity of MSCs in M1 macrophage-driven inflammation. The insights into the challenges and how they were addressed may also be helpful to developers of potency assays related to other cellular functions and clinical indications.

Optimization and Validation of a Harmonized Protocol for Generating Therapeutic-Grade Dendritic Cells in a Randomized Phase II Clinical Trial, Using Two Varied Antigenic Sources.

Autologous dendritic cell (DC)-based immunotherapy is a cell-based advanced therapy medicinal product (ATMP) that was first introduced more than three decades ago. In the current study, our objective was to establish a harmonized protocol using two varied antigenic sources and a good manufacturing practice (GMP)-compliant, manual method for generating clinical-grade DCs at a limited-resource academic setting. After obtaining ethical committee-approved informed consent, the recruited patients underwent leukapheresis, and single-batch DC production was carried out. Using responder-independent flow cytometric assays as quality control (QC) criteria, we propose a differentiation and maturation index (DI and MI, respectively), calculated with the QC cut-off and actual scores of each batch for comparison. Changes during cryopreservation and personnel variation were assessed periodically for up to two to three years. Using our harmonized batch production protocol, the average DI was 1.39 and MI was 1.25. Allogenic responder proliferation was observed in all patients, while IFN-gamma secretion, evaluated using flow cytometry, was detected in 10/36 patients and significantly correlated with CD8+ T cell proliferation (p value-0.0002). Tracking the viability and phenotype of cryopreserved MDCs showed a >90% viability for up to three years, while a mature DC phenotype was retained for up to one year. Our results confirm that the manual/semi-automated protocol was simple, consistent, and cost-effective, without the requirement for expensive equipment and without compromising on the quality of the final product.

Osmotic properties of T cells determined by flow imaging microscopy in comparison to electrical sensing zone analysis

To develop cryopreservation methods for cell-based medicinal products it is important to understand osmotic responses of cells upon immersion into solutions with cryoprotective agents (CPAs) and during freezing. The aim of this study was to assess the osmotic response of T cells by using flow imaging microscopy (FIM) as a novel cell-sizing technique, and to corroborate the findings with electrical impedance measurements conducted on a Coulter counter. Jurkat cells were used as a potential model cell line for primary T cells. Cell volume responses were used to derive important cell parameters for cryopreservation such as the osmotically inactive cell volume Vb and the membrane permeability towards water and various CPAs. Unlike Coulter counter measurement, FIM, combined with Trypan blue staining can differentiate between viable and dead cells, which yields a more accurate estimation of Vb. Membrane permeabilities to water, dimethyl sulfoxide (Me2SO) and glycerol were measured for Jurkat cells at different temperatures. The permeation of Me2SO into the cells was faster in comparison to glycerol. CPA permeation decreased with decreasing temperature following Arrhenius behavior. Moreover, membrane permeability to water decreased in the presence of CPAs. Vb of Jurkat cells was found to be 49% of the isotonic volume and comparable to that of primary T cells. FIM proved to be a valuable tool to determine the membrane permeability parameters of mammalian cells to water and cryoprotective agents, which in turn can be used to rationally design CPA loading procedures for cryopreservation.

Cell-based medicinal products approved in the European Union: current evidence and perspectives.

Advanced Therapy Medicinal Products (ATMPs) are innovative clinical treatments exploiting the pharmacological, immunological, or metabolic properties of cells and/or gene(s) with the aim to restore, correct, or modify a biological function in the recipient. ATMPs are heterogeneous medicinal products, developed mainly as individualized and patient-specific treatments, and represent new opportunities for diseases characterized by a high-unmet medical need, including rare, genetic and neurodegenerative disorders, haematological malignancies, cancer, autoimmune, inflammatory and orthopaedic conditions. Into the European Union (EU) market, the first ATMP has been launched in 2009 and, to date, a total of 24 ATMPs have been approved. This review aims at reporting on current evidence of cell-based therapies authorized in the EU, including Somatic Cell Therapies, Tissue Engineering Products, and Cell-based Gene Therapy Products as Chimeric Antigen Receptor (CAR) T-cells, focusing on the evaluation of efficacy and safety in clinical trials and real-world settings. Despite cell-based therapy representing a substantial promise for patients with very limited treatment options, some limitations for its widespread use in the clinical setting remain, including restricted indications, highly complex manufacturing processes, elevated production costs, the lability of cellular products over time, and the potential safety concerns related to the intrinsic characteristics of living cells, including the risk of severe or life-threatening toxicities, such as CAR-T induced neurotoxicity and cytokine release syndrome (CRS). Although encouraging findings support the clinical use of ATMPs, additional data, comparative studies with a long-term follow-up, and wider real-world evidences are needed to provide further insights into their efficacy and safety profiles.

Clinical Trials of Cellular Therapies in Solid Tumors.

In the past years cancer treatments have drastically changed, mainly due to the development of immune checkpoint inhibitors capable of immune modulation in vivo, thus providing major clinical benefit in a number of malignancies. Simultaneously, considerable technical refinements have opened new prospects for the development of immune cell-based medicinal products and unprecedented success with chimeric antigen receptor (CAR)-T cells targeting B-cell hematologic malignancies has been obtained. However, T cell therapies introduced and performed in the field of solid tumors have produced so far only limited responses in selected patient populations. This standstill is attributable to the difficulty in identifying target antigens which are homogeneously expressed by all tumor cells while absent from normal tissues, and the limited T cell persistence and proliferation in a hostile tumor microenvironment that favors immune escape. Replicating the results observed in hematology is a major scientific challenge in solid tumors, and ongoing translational and clinical research is focused on obtaining insight into the mechanisms of tumor recognition and evasion, and how to improve the efficacy of cellular therapies, also combining them with immune checkpoint inhibitors or other agents targeting either the cancer cell or the tumor environment. This paper provides an overview of current adaptive T cell therapy approaches in solid tumors, the research performed to increase their efficacy and safety, and results from ongoing clinical trials.

Potency assays and biomarkers for cell-based advanced therapy medicinal products.

Advanced Therapy Medicinal Products (ATMPs) based on somatic cells expanded in vitro, with or without genetic modification, is a rapidly growing area of drug development, even more so following the marketing approval of several such products. ATMPs are produced according to Good Manufacturing Practice (GMP) in authorized laboratories. Potency assays are a fundamental aspect of the quality control of the end cell products and ideally could become useful biomarkers of efficacy in vivo. Here we summarize the state of the art with regard to potency assays used for the assessment of the quality of the major ATMPs used clinic settings. We also review the data available on biomarkers that may substitute more complex functional potency tests and predict the efficacy in vivo of these cell-based drugs.

Current approaches to quality assessment, non-clinical and clinical studies of dendritic cell vaccines in oncology

At present, personalised cellular immunotherapy is considered a promising approach to the treatment of malignant neoplasms. The effectiveness of these cellular immunotherapy methods is evaluated in the context of clinical and biological tumour characteristics and the state of the immune system of a particular patient. One of the immunotherapy options for cancer is the development of autologous dendritic cell vaccines.The aim of this study was to analyse current methodological approaches to the evaluation of the quality, efficacy, and safety of dendritic cell cancer vaccines.This review describes the functional role of dendritic cells in immune response regulation. The paper presents the results of literature analysis covering current approaches to obtaining dendritic cell vaccines with specific characteristics, quality assessment, studies of the anti-tumour efficacy of cell therapy products, and the experience of conducting non-clinical and clinical studies. The review highlights specific aspects of international experience in the registration and clinical use of cell therapy products. The authors discuss methodological approaches to non-clinical studies of dendritic cell vaccines, which should aim to obtain information to select the dose, route, and mode of administration and to identify immunological markers correlating to the clinical efficacy of cell therapy products. The paper covers international experience in conducting clinical trials of dendritic cell vaccines for various malignant neoplasms. The authors propose a list of quality attributes of human somatic cell-based medicinal products for further clinical use.

Microbiological Aspects of Pharmaceutical Manufacturing of Adipose-Derived Stem Cell-Based Medicinal Products.

Subcutaneous adipose tissue is an excellent source of mesenchymal stem cells (ADSCs), which can be used in cell therapies as an active substance in advanced therapy medicinal products (ATMPs). Because of the short shelf-life of ATMPs and the time needed to obtain the results of microbiological analysis, the final product is often administered to the patient before sterility is confirmed. Because the tissue used for cell isolation is not sterilized to maintain cell viability, controlling and ensuring microbiological purity at all stages of production is crucial. This study presents the results of monitoring the contamination incidence during ADSC-based ATMP manufacturing over two years. It was found that more than 40% of lipoaspirates were contaminated with thirteen different microorganisms, which were identified as being physiological flora from human skin. Such contamination was successfully eliminated from the final ATMPs through the implementation of additional microbiological monitoring and decontamination steps at various stages of production. Environmental monitoring revealed incidental bacterial or fungal growth, which did not result in any product contamination and was reduced thanks to an effective quality assurance system. To conclude, the tissue used for ADSC-based ATMP manufacturing should be considered contaminated; therefore, good manufacturing practices specific to this type of product must be elaborated and implemented by the manufacturer and the clinic in order to obtain a sterile product.

Brain Organoids to Evaluate Cellular Therapies.

Animal models currently used to test the efficacy and safety of cell therapies, mainly murine models, have limitations as molecular, cellular, and physiological mechanisms are often inherently different between species, especially in the brain. Therefore, for clinical translation of cell-based medicinal products, the development of alternative models based on human neural cells may be crucial. We have developed an in vitro model of transplantation into human brain organoids to study the potential of neural stem cells as cell therapeutics and compared these data with standard xenograft studies in the brain of immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Neural stem cells showed similar differentiation and proliferation potentials in both human brain organoids and mouse brains. Our results suggest that brain organoids can be informative in the evaluation of cell therapies, helping to reduce the number of animals used for regulatory studies.

Engineering bone-forming callus organoid implants in a xenogeneic-free differentiation medium

The field of tissue engineering aspires to provide clinically relevant solutions for patients through the integration of developmental engineering principles with a bottom-up manufacturing approach. However, the manufacturing of cell-based advanced therapy medicinal products is hampered by protocol complexity, lack of non-invasive critical quality controls, and dependency on animal-derived components for tissue differentiation. We investigate a serum-free, chemically defined, xeno- and lipid-free chondrogenic differentiation medium to generate bone-forming callus organoids. Our results show an increase in microtissue homogeneity during prolonged differentiation and the high quality of in vivo bone-forming organoids. The low protein content of the culture medium potentially allows for the monitoring of relevant secreted biomarkers as (critical) quality attributes. Together, we envisage that this xeno- and lipid-free chondrogenic medium is compatible with industrial scale-up and automation while facilitating the implementation of non-invasive imaging and the use of quality control parameters based on secreted biomarkers.

Optimization of human platelet lysate production and pathogen reduction in a public blood transfusion center.

Human platelet lysate (HPL) has been proposed as a safe and efficient xeno-free alternative to fetal bovine serum (FBS) for large-scale culturing of cell-based medicinal products. However, the use of blood derivatives poses a potential risk of pathogen transmission. To mitigate this risk, different pathogen reduction treatment (PRT) practices can be applied on starting materials or on final products, but these methods might modify the final composition and the quality of the products. We evaluated the impact of applying a PRT based on riboflavin and ultraviolet irradiation on the raw materials used to manufacture an improved Good Manufacturing Practices (GMP)-grade HPL product in a public blood center. Growth promotion and the levels of growth factors and proteins were compared between an inactivated product (HPL4-i) and a non-inactivated product (HPL4). Stability studies were performed at 4°C, -20°C, and -80°C. The application of a PRT on the starting materials significantly altered the protein composition of HPL4-i as compared with HPL4. Despite this, the growth promoting rates were unaffected when compared with FBS used as a control. While all products were stable at -20°C and -80°C for 24 months, a significant decrease in the activity of HPL4-i was observed when stored at 4°C. Our results show that the application of a PRT based on riboflavin and ultraviolet light on starting materials used in the manufacture of HPL modifies the final composition of the product, yet its cell growth promoting activity is maintained at levels similar to those of non-inactivated products.

Development of a human umbilical cord-derived mesenchymal stromal cell-based advanced therapy medicinal product to treat immune and/or\xa0inflammatory diseases

BackgroundUmbilical cord-derived mesenchymal stromal cells (UC-MSCs) revealed their key role in immune regulation, offering promising therapeutic perspectives for immune and inflammatory diseases. We aimed to develop a production process of an UC-MSC-based product and then to characterize UC-MSC properties and immunomodulatory activities in vitro, related to their clinical use and finally, to transfer this technology to a good manufacturing practice (GMP) compliant facility, to manufacture an advanced therapy medicinal product (ATMP).MethodsFifteen human umbilical cords (UCs) were collected to develop the production process. Three batches of UC-MSCs from a single donor were characterized at basal state and after in vitro pro-inflammatory stimulation by interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα). Proliferation, immunophenotype, activation markers’ expression and the inhibition of T cell proliferation were assessed. Finally, this technology was transferred to a GMP-compliant facility to manufacture an UC-MSC-based ATMP, from a single donor, using the explant method followed by the establishment of master and work cell stocks.ResultsTwelve UCs were processed successfully allowing to isolate UC-MSCs with doubling time and population doubling remaining stable until passage 4. CD90, CD105, CD73, CD44, CD29, CD166 expression was positive; CD14, CD45, CD31, HLA-DR, CD40, CD80 and CD86 expression was negative, while CD146 and HLA-ABC expression was heterogeneous. Cell morphology, proliferation and immunophenotype were not modified by inflammatory treatment. Indoleamine 2,3-dioxygenase (IDO) expression was significantly induced by IFNγ and IFNγ + TNFα versus non-treated cells. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression was induced significantly after priming. T cell proliferation was significantly decreased in the presence of UC-MSCs in a dose-dependent manner. This inhibitory effect was improved by IFNγ or IFNγ + TNFα, at UC-MSCs:PBMC ratio 1:10 and 1:30, whereas only IFNγ allowed to decrease significantly T cell proliferation at ratio 1:100. The manufacturing process of the UC-MSC-based ATMP was qualified and authorized by the French regulatory agency for clinical use (NCT04333368).ConclusionThis work allowed to develop an investigational UC-MSC-based ATMP authorized for clinical use. Our results showed that an inflammatory environment preserves the biological properties of UC-MSCs with an improvement of their immunomodulatory functions.

Human Neural Stem Cells for Cell-Based Medicinal Products.

Neural stem cells represent an attractive tool for the development of regenerative therapies and are being tested in clinical trials for several neurological disorders. Human neural stem cells can be isolated from the central nervous system or can be derived in vitro from pluripotent stem cells. Embryonic sources are ethically controversial and other sources are less well characterized and/or inefficient. Recently, isolation of NSC from the cerebrospinal fluid of patients with spina bifida and with intracerebroventricular hemorrhage has been reported. Direct reprogramming may become another alternative if genetic and phenotypic stability of the reprogrammed cells is ensured. Here, we discuss the advantages and disadvantages of available sources of neural stem cells for the production of cell-based therapies for clinical applications. We review available safety and efficacy clinical data and discuss scalability and quality control considerations for manufacturing clinical grade cell products for successful clinical application.

Forced degradation of cell-based medicinal products guided by flow imaging microscopy: Explorative studies with Jurkat cells

Cell-based medicinal products (CBMPs) offer ground-breaking opportunities to treat diseases with limited or no therapeutic options. However, the intrinsic complexity of CBMPs results in great challenges with respect to analytical characterization and stability assessment. In our study, we submitted Jurkat cell suspensions to forced degradation studies mimicking conditions to which CBMPs might be exposed from procurement of cells to administration of the product. Flow imaging microscopy assisted by machine learning was applied for determination of cell viability and concentration, and quantification of debris particles. Additionally, orthogonal cell characterization techniques were used. Thawing of cells at 5 °C was detrimental to cell viability and resulted in high numbers of debris particles, in contrast to thawing at 37 °C or 20 °C which resulted in better stability. After freezing of cell suspensions at −18 °C in presence of dimethyl sulfoxide (DMSO), a DMSO concentration of 2.5% (v/v) showed low stabilizing properties, whereas 5% or 10% was protective. Horizontal shaking of cell suspensions did not affect cell viability, but led to a reduction in cell concentration. Fetal bovine serum (10% [v/v]) protected the cells during shaking. In conclusion, forced degradation studies with application of orthogonal analytical characterization methods allow for CBMP stability assessment and formulation screening.

Mesangiogenic Progenitor Cells Are Tissue Specific and Cannot Be Isolated From Adipose Tissue or Umbilical Cord Blood.

Mesangiogenic progenitor cells (MPCs) have been isolated from human bone marrow (BM) mononuclear cells. They attracted particular attention for the ability to differentiate into exponentially growing mesenchymal stromal cells while retaining endothelial differentiative potential. MPC power to couple mesengenesis and angiogenesis highlights their tissue regenerative potential and clinical value, with particular reference to musculoskeletal tissues regeneration. BM and adipose tissue represent the most promising adult multipotent cell sources for bone and cartilage repair, although discussion is still open on their respective profitability. Culture determinants, as well as tissues of origin, appeared to strongly affect the regenerative potential of cell preparations, making reliable methods for cell isolation and growth a prerequisite to obtain cell-based medicinal products. Our group had established a definite consistent protocol for MPC culture, and here, we present data showing MPCs to be tissue specific.