Emerging technologies for quality control of cell-based, advanced therapy medicinal products.

Advanced therapy medicinal products (ATMP) are complex medicines based on gene therapy, somatic cell therapy, and tissue engineering. These products are rapidly arising as novel and promising therapies for a wide range of different clinical applications. The process for the development of well-established ATMPs is challenging. Many issues must be considered from raw material, manufacturing, safety, and pricing to assure the quality of ATMPs and their implementation as innovative therapeutic tools. Among ATMPs, cell-based ATMPs are drugs altogether. As for standard drugs, technologies for quality control, and non-invasive isolation and production of cell-based ATMPs are then needed to ensure their rapidly expanding applications and ameliorate safety and standardization of cell production. In this review, emerging approaches and technologies for quality control of innovative cell-based ATMPs are described. Among new techniques, microfluid-based systems show advantages related to their miniaturization, easy implementation in analytical process and automation which allow for the standardization of the final product.

Urine-Derived Renal Epithelial Cells (URECs) from Transplanted Kidneys as a Promising Immunomodulatory Cell Population.

Kidney transplantation is a lifesaving procedure for patients with end-stage kidney disease (ESKD). Organs derived from donation after cardiac death (DCD) are constantly increasing; however, DCD often leads to ischaemia-reperfusion (IR) and Acute Kidney Injury (AKI) events. These phenomena increase kidney cell turnover to replace damaged cells, which are voided in urine. Urine-derived renal epithelial cells (URECs) are rarely present in the urine of healthy subjects, and their loss has been associated with several kidney disorders. The present study aimed to characterize the phenotype and potential applications of URECs voided after transplant. The results indicate that URECs are highly proliferating cells, expressing several kidney markers, including markers of kidney epithelial progenitor cells. Since the regulation of the immune response is crucial in organ transplantation and new immunoregulatory strategies are needed, UREC immunomodulatory properties were investigated. Co-culture with peripheral blood mononuclear cells (PBMCs) revealed that URECs reduced PBMC apoptosis, inhibited lymphocyte proliferation, increased T regulatory (Treg) cells and reduced T helper 1 (Th1) cells. URECs from transplanted patients represent a promising cell source for the investigation of regenerative processes occurring in kidneys, and for cell-therapy applications based on the regulation of the immune response.

Mitigating Oxidative Stress in Perinatal Cells: A Critical Step toward an Optimal Therapeutic Use in Regenerative Medicine.

Oxidative stress (OS) occurs when the production of reactive oxygen species (ROS) is not balanced by the body's antioxidant defense system. OS can profoundly affect cellular health and function. ROS can have a profound negative impact on cells that undergo a predestined and time-regulated process of proliferation or differentiation, such as perinatal stem cells. Due to the large-scale employment of these immunotolerant stem cells in regenerative medicine, it is important to reduce OS to prevent them from losing function and increase their application in the regenerative medicine field. This goal can be achieved through a variety of strategies, such as the use of antioxidants and other compounds that can indirectly modulate the antioxidant defense system by enhancing cellular stress response pathways, including autophagy and mitochondrial function, thereby reducing ROS levels. This review aims to summarize information regarding OS mechanisms in perinatal stem cells and possible strategies for reducing their deleterious effects.

Characterization of Perinatal Stem Cell Spheroids for the Development of Cell Therapy Strategy.

Type 1 diabetes mellitus (T1DM) is a complex metabolic disease characterized by a massive loss of insulin-producing cells due to an autoimmune reaction. Currently, daily subcutaneous administration of exogenous insulin is the only effective treatment. Therefore, in recent years considerable interest has been given to stem cell therapy and in particular to the use of three-dimensional (3D) cell cultures to better reproduce in vivo conditions. The goal of this study is to provide a reliable cellular model that could be investigated for regenerative medicine applications for the replacement of insulin-producing cells in T1DM. To pursue this aim we create a co-culture spheroid of amniotic epithelial cells (AECs) and Wharton's jelly mesenchymal stromal cells (WJ-MSCs) in a one-to-one ratio. The resulting co-culture spheroids were analyzed for viability, extracellular matrix production, and hypoxic state in both early- and long-term cultures. Our results suggest that co-culture spheroids are stable in long-term culture and are still viable with a consistent extracellular matrix production evaluated with immunofluorescence staining. These findings suggest that this co-culture may potentially be differentiated into endo-pancreatic cells for regenerative medicine applications in T1DM.

The Medium Obtained from the Culture of Hodgkin Lymphoma Cells Affects the Biophysical Characteristics of a Fibroblast Cell Model.

The neoplastic Hodgkin-Reed-Sternberg (HRS) cells in Hodgkin lymphoma (HL) represent only 1-10% of cells and are surrounded by an inflammatory microenvironment. The HL cytokine network is a key point for the proliferation of HRS cells and for the maintenance of an advantageous microenvironment for HRS survival. In the tumor microenvironment (TME), the fibroblasts are involved in crosstalk with HRS cells. The aim of this work was to study the effect of lymphoma cell conditioned medium on a fibroblast cell population and evaluate modifications of cell morphology and proliferation. Hodgkin lymphoma-derived medium was used to obtain a population of "conditioned" fibroblasts (WS-1 COND). Differences in biophysical parameters were detected by the innovative device Celector®. Fibroblast-HL cells interactions were reproduced in 3D co-culture spheroids. WS-1 COND showed a different cellular morphology with an enlarged cytoplasm and enhanced metabolism. Area and diameter cell values obtained by Celector® measurement were increased. Co-culture spheroids created with WS-1 COND showed a tighter aggregation than those with non-conditioned WS-1. The presence of soluble factors derived from HRS cells in the conditioned medium was adequate for the proliferation of fibroblasts and conditioned fibroblasts in a 3D HL model allowed to develop a representative model of the in vivo TME.

Perinatal Stem Cell Therapy to Treat Type 1 Diabetes Mellitus: A Never-Say-Die Story of Differentiation and Immunomodulation.

Human term placenta and other postpartum-derived biological tissues are promising sources of perinatal cells with unique stem cell properties. Among the massive current research on stem cells, one medical focus on easily available stem cells is to exploit them in the design of immunotherapy protocols, in particular for the treatment of chronic non-curable human diseases. Type 1 diabetes is characterized by autoimmune destruction of pancreatic beta cells and perinatal cells can be harnessed both to generate insulin-producing cells for beta cell replenishment and to regulate autoimmune mechanisms via immunomodulation capacity. In this study, the strong points of cells derived from amniotic epithelial cells and from umbilical cord matrix are outlined and their potential for supporting cell therapy development. From a basic research and expert stem cell point of view, the aim of this review is to summarize information regarding the regenerative medicine field, as well as describe the state of the art on possible cell therapy approaches for diabetes.

LFA-1 Controls Th1 and Th17 Motility Behavior in the Inflamed Central Nervous System.

Leukocyte trafficking is a key event during autoimmune and inflammatory responses. The subarachnoid space (SAS) and cerebrospinal fluid are major routes for the migration of encephalitogenic T cells into the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis, and are sites of T cell activation before the invasion of CNS parenchyma. In particular, autoreactive Th1 and Th17 cell trafficking and reactivation in the CNS are required for the pathogenesis of EAE. However, the molecular mechanisms controlling T cell dynamics during EAE are unclear. We used two-photon laser microscopy to show that autoreactive Th1 and Th17 cells display distinct motility behavior within the SAS in the spinal cords of mice immunized with the myelin oligodendrocyte glycoprotein peptide MOG35-55. Th1 cells showed a strong directional bias at the disease peak, moving in a straight line and covering long distances, whereas Th17 cells exhibited more constrained motility. The dynamics of both Th1 and Th17 cells were strongly affected by blocking the integrin LFA-1, which interfered with the deformability and biomechanics of Th1 but not Th17 cells. The intrathecal injection of a blocking anti-LFA-1 antibody at the onset of disease significantly inhibited EAE progression and also strongly reduced neuro-inflammation in the immunized mice. Our results show that LFA-1 plays a pivotal role in T cell motility during EAE and suggest that interfering with the molecular mechanisms controlling T cell motility can help to reduce the pathogenic potential of autoreactive lymphocytes.

Prevalence of chronic pancreatitis: Results of a primary care physician-based population study.

BACKGROUND: Data on chronic pancreatitis prevalence are scanty and usually limited to hospital-based studies. AIM: Investigating chronic pancreatitis prevalence in primary care. METHODS: Participating primary care physicians reported the prevalence of chronic pancreatitis among their registered patients, environmental factors and disease characteristics. The data were centrally reviewed and chronic pancreatitis cases defined according to M-ANNHEIM criteria for diagnosis and severity and TIGAR-O classification for etiology. RESULTS: Twenty-three primary care physicians participated in the study. According to their judgment, 51 of 36.401 patients had chronic pancreatitis. After reviewing each patient data, 11 turned out to have definite, 5 probable, 19 borderline and 16 uncertain disease. Prevalence was 30.2/100.000 for definite cases and 44.0/100.000 for definite plus probable cases. Of the 16 patients with definite/probable diagnosis, 8 were male, with mean age of 55.6 (±16.7). Four patients had alcoholic etiology, 5 post-acute/recurrent pancreatitis, 6 were deemed to be idiopathic. Four had pancreatic exocrine insufficiency, 10 were receiving pancreatic enzymes, and six had pain. Most patients had initial stage and non-severe disease. CONCLUSIONS: This is the first study investigating the prevalence of chronic pancreatitis in primary care. Results suggest that the prevalence in this context is higher than in hospital-based studies, with specific features, possibly representing an earlier disease stage.