18^th Global Summit on Stem Cell & Regenerative Medicine November 09-10, 2022 Rome, Italy

Biography:

Abstract:

Biography:

Abstract:

Biography:

Abstract:

Biography:

Abstract:

Biography:

Nishant Joglekar is a final year doctoral researcher from Loughborough University, UK. His PhD is focused on addressing the effects of cryopreservation during the manufacturing of stem cell-based cell therapies, developing a rapid method to identify biomarkers that could be used to detect dying cells, post-thaw, during manufacturing. Nishant is skilled in cell culture (T cells and stem cells), and numerous other cell-based techniques including flow cytometry, qPCR, and fluorescence microscopy.

Abstract:

Statement of the Problem: There is currently a growing interest in the use of Mesenchymal Stem Cells (MSCs) in the development of cell therapies for the treatment of many common conditions. However, during manufacturing, apoptosis can lead to a loss of function following cryopreservation and remains an area of concern. Delayed apoptosis in which cells that appear viable immediately post-thaw undergo apoptosis in a delayed manner is a particular challenge, with no rapid method to identify such cell populations at an early stage. The aim of this work is to develop a rapid method, using metabolomics, to detect biomarkers, post-thaw, that could indicate cells undergoing apoptosis. Methodology & Theoretical Orientation: Initially, cells were cryopreserved using different freezing procedures expected to have varying impacts on the cells post-thaw (PT). These included changing the amount of DMSO concentration and freezing rate. The proliferation and viability of the cells, PT, across five days was then investigated, along with the immunomodulatory properties of MSCs PT, including their ability to reduce T cell proliferation and induce T cell differentiation into T regulatory cells (Tregs). Liquid Chromatography / Mass Spectrometry (LC/MS), and Gas Chromatography / Mass Spectrometry (LC/MS) are now being used to detect apoptotic biomarkers, PT, that could give an indication of cells undergoing apoptosis. Findings: Using detrimental freezing procedures showed a significant decrease in viability and the proliferation of the cells PT. However, cryopreservation did not affect the immunomodulatory properties of the MSCs. Initial preliminary work using GC/MS has shown that healthy MSCs reduce the amount of a specific compound in the headspace of cell cultures over 24hrs PT. Conclusion & Significance: While results are only preliminary, the decrease in the compound, PT, could be used as a biomarker for cell health. This could be used as a rapid quality control step during manufacturing.

Biography:

Miriama Sikorová is currently a student of the 2nd year of doctoral studies at the 1st Medical Faculty of Charles University in Prague, in the field of Cell Biology and Pathobiology. She has started her research career while studying at university, where she became enthused for molecular, but also immunological and cytological research. She is currently engaged in studies of microenvironment of leukemia cells. Cell cultures and their biochemical, molecular, cytological, microscopic, and no less important statistical analyses take significant proportion of her time. She always tries to research as deeply as possible, in order to understand broader context. Thanks to her own intense interest, as well as an active approach, she constantly advances in acquired theoretical and practical knowledge within the given issue.

Abstract:

Preceding researches of hematological malignancies have focused their research on Patient-derived xenograft models when directly insert patient tumor cells into immunodeficient mice. Culture conditions and other factors presented in the leukemic microenvironment don´t reflect the in vivo situation and may control behavior of the cultivated leukemic cells resulting in decrease of their viability and influencing other attributes too. We aim our project to conquer these drawbacks by testing new approaches for leukemic cell in vitro cultivation comprising inspection of metabolic, proliferative and morphological changes of several leukemic cell types.

As feeder cells we used human dermal fibroblasts (NHDF) and human mesenchymal stem cells. As leukemic cells HBL-2 cell line (Human Mantle Cell Lymphoma); SD-1 cell line (acute lymphoblastic leukemia) and UPF 4D cell line (Diffuse large B‑cell lymphoma) were used. Our experiments focused on a single cell cultivation as well as a co-cultivation of selected leukemic and feeder cells performed simultaneously under hypoxic (1% O2) and normoxic (20% O2) conditions. Comprehensive examination of all types of leukemic cells revealed differences in number of cells and a type of energy metabolism, leaning on the form of cultivation, type of used feeder cells as well as on oxygen concentration.

Generally, the leukemic cell lines more proliferated in co-cultivation with both feeders, however, the oxygen concentration was decisive. In co-cultivation with NHDF feeder cells, HBL-2 cells´ proliferation was significantly increased only in hypoxia, while SD-1 cells´ only in normoxia. In case of UPF 4 D cells, hypoxia increased their proliferation in co-cultivation with both types of feeders. The type of energy metabolism of leukemic cells changed in favor of glycolysis with decreasing oxygen concentration. In summary, our findings might be an acquisition for further improvement of the character of leukemic cells in vitro for their subsequent usage in successful in vivo xenotransplantation.

Biography:

Kaixuan Zhao is a PhD candidate in Medical Science at Karolinska Institutet, Sweden. She is studying the role of voltage-gated Ca²⁺ channels in beta cell maturity. Her efforts have resulted in interesting publications in Proc Natl Acad Sci USA, Cell Mol Life Sci and Cell Transplantat.

Abstract:

T-type Ca²⁺ channels operate in embryonic stem cells, but conduct relatively small Ca²⁺ currents in mature human β cells. In certain pathological contexts, e.g., when T-type Ca²⁺ channels undergo elevated expression, they mediate exaggerated Ca²⁺ influx to dissipate β cell maturity. This prompted us to hypothesize that altered T-type Ca²⁺ channel activity in human iPSC-derived islet (hiPSC-islet) cells affect maturation. To test our hypothesis, we transplanted hiPSC-isles into the anterior chamber of the eye (ACE) of immunodeficient mice, intravitreally infused T-type Ca²⁺ channel blocker NNC55-0396 and performed in vivo and ex vivo measurements. In vivo stereomicroscopy showed that transplanted hiPSC-islets underwent initial adhesion to, gradual integration with and eventual engraftment as well as survival on the iris. In vivo confocal microscopy revealed that intracameral hiPSC-islets were satisfactorily vascularized and displayed intense light scattering signals, reflecting the abundance of zinc-insulin crystals inside insulin secretory granules, within two months post-transplantation. Furthermore, intravitreally-infused NNC55-0396 did not influence the macromorphology, vascularization and light scattering signals. Interestingly, ex vivo [Ca²⁺]_i measurements disclosed that intravitreally-infused NNC55-0396 significantly decreased basal [Ca²⁺]_i levels and increased glucose-stimulated [Ca²⁺]_i responses in intact hiPSC-isles. In conclusion, the present study verifies that the immunodeficient mouse ACE can serve as a unique site for pharmacological manipulation of in vivo maturation of hiPSC-islets. These cells can not only be micro-imaged intravitally, noninvasively and longitudinally, but also retrieved without suffering physical and chemical disturbance for more precise ex vivo studies, as exemplified here by [Ca²⁺]_i measurements. Importantly, our data demonstrate that inhibition of T-Type Ca²⁺ channels facilitates glucose-dependent Ca²⁺ signaling in hiPSC-islets. These findings are important and support the notion that altered T-type Ca²⁺ channel activity may serve as a key signal in hiPSC-islet cell maturation.

Biography:

Idiberto Jose Zotarelli Filho  is affiliated from ABRAN- Brazilian Association of Nutrology, Brazil.. He is a recipient of many awards and grants for his valuable contributions and discoveries in major area of subject research. His international experience includes various programs, contributions and participation in different countries for diverse fields of study.  His research interests reflect in his wide range of publications in various national and international journals.  Keyword Adipose mesenchymal stem cells

Abstract:

Introduction: Therapy of artificial tissues and organs has increased the life expectancy of thousands of people, and one in five over 65 years has already benefited from tissues and organs generated in vitro. Thus, it is imperative to develop new strategies for the development and optimization of biomatrices and stem cells to meet demand, which is capable of promoting tissue and organ regeneration, cost amortization, and safe use at the cellular level. In this context, adipose mesenchymal stem cells (AMSC) have aroused great scientific interest. Objective: The biocompatibility at the cellular and chromosomal/chromatidic level of chitosan-collagen-genipin biomatrix with human AMSC was analyzed. Methods: Different concentrations of genipin for matrix establishment were tested by the analysis of the number of cells, the degradation, and the degree of crosslinking by spectrophotometry. AMSC was obtained by enzymatic processing of human lipoaspirate and cultured in Petakas (control) and the selected biomatrix after physicochemical characterization, with and without cryopreservation. The AMSC were characterized for adhesion and proliferation by conventional optical microscopy, phase contrast and scanning electron microscopy, immunophenotyping, cell viability with Trypan blue test, chromosomal stability, by conventional cytogenetic techniques, neoplastic potential by Papanicolaou test, and the capacity of cell differentiation in three types of tissues, with a statistical approach. Results: The crosslinked biomatrix with 0.75% v/v genipin was the best for the cell studies. The tests of adhesion, proliferation, and differentiation capacity showed that the AMSC in contact with the biomatrix maintained their natural characteristics. The cytogenetic and Papanicolaou tests after different culture conditions in the biomatrix did not reveal statistically different frequencies of the control cells (cultured without the biomatrix). Conclusion: The results suggest that the chitosan-collagen-genipin biomatrices with 0.75% v/v genipin are biocompatible at the cellular and chromatidic/chromosomal level, with adipose mesenchymal stem cells.