Adipose-derived stem cells (ASCs) culture in spinner flask: improving the parameters of culture in a microcarrier-based system.

Prior to clinical use, extensive in vitro proliferation of human adipose-derived stem cells (ASCs) is required. Among the current options, spinner-type stirred flasks, which use microcarriers to increase the yield of adherent cells, are recommended. Here, we propose a methodology for ASCs proliferation through cell suspension culture using Cultispher-S® microcarriers (MC) under agitation in a spinner flask, with the aim of establishing a system that reconciles the efficiency of cell yield with high viability of the culture during two distinct phases: seeding and proliferation. The results showed that cell adhesion was potentiated under intermittent stirring at 70 rpm in the presence of 10% FBS for an initial cell concentration of 2.4 × 104 cells/mL in the initial 24 h of cultivation. In the proliferation phase, kinetic analysis showed that cell growth was higher under continuous agitation at 50 rpm with a culture medium renewal regime of 50% every 72 h, which was sufficient to maintain the culture at optimal levels of nutrients and metabolites for up to nine days of cultivation, representing an 11.1-fold increase and a maximum cell productivity of 422 cells/mL/h (1.0 × 105 viable cells/mL). ASCs maintained the immunophenotypic characteristics and mesodermal differentiation potential of both cell lines from different donors. The established protocol represents a more efficient and cost-effective method to obtain a high proliferation rate of ASCs in a microcarrier-based system, which is necessary for large-scale use in cell therapy, highlighting that the manipulation of critical parameters optimizes the ASCs production process.

Adipose-derived stem cells (ASCs) culture in spinner flask: improving the parameters of culture in a microcarrier-based system

Prior to clinical use, extensive in vitro proliferation of human adipose-derived stem cells (ASCs) is required. Among the current options, spinner-type stirred flasks, which use microcarriers to increase the yield of adherent cells, are recommended. Here, we propose a methodology for ASCs proliferation through cell suspension culture using Cultispher-S® microcarriers (MC) under agitation in a spinner flask, with the aim of establishing a system that reconciles the efficiency of cell yield with high viability of the culture during two distinct phases: seeding and proliferation. The results showed that cell adhesion was potentiated under intermittent stirring at 70 rpm in the presence of 10% FBS for an initial cell concentration of 2.4 × 104 cells/mL in the initial 24 h of cultivation. In the proliferation phase, kinetic analysis showed that cell growth was higher under continuous agitation at 50 rpm with a culture medium renewal regime of 50% every 72 h, which was sufficient to maintain the culture at optimal levels of nutrients and metabolites for up to nine days of cultivation, representing an 11.1-fold increase and a maximum cell productivity of 422 cells/mL/h (1.0 × 105 viable cells/mL). ASCs maintained the immunophenotypic characteristics and mesodermal differentiation potential of both cell lines from different donors. The established protocol represents a more efficient and cost-effective method to obtain a high proliferation rate of ASCs in a microcarrier-based system, which is necessary for large-scale use in cell therapy, highlighting that the manipulation of critical parameters optimizes the ASCs production process.

Combination of genetically engineered T cells and immune checkpoint blockade for the treatment of cancer.

Immune checkpoint (IC) blockade using monoclonal antibodies is currently one of the most successful immunotherapeutic interventions to treat cancer. By reinvigorating antitumor exhausted T cells, this approach can lead to durable clinical responses. However, the majority of patients either do not respond or present a short-lived response to IC blockade, in part due to a scarcity of tumor-specific T cells within the tumor microenvironment. Adoptive transfer of T cells genetically engineered to express chimeric antigen receptors (CARs) or engineered T-cell receptors (TCRs) provide the necessary tumor-specific immune cell population to target cancer cells. However, this therapy has been considerably ineffective against solid tumors in part due to IC-mediated immunosuppressive effects within the tumor microenvironment. These limitations could be overcome by associating adoptive cell transfer of genetically engineered T cells and IC blockade. In this comprehensive review, we highlight the strategies and outcomes of preclinical and clinical attempts to disrupt IC signaling in adoptive T-cell transfer against cancer. These strategies include combined administration of genetically engineered T cells and IC inhibitors, engineered T cells with intrinsic modifications to disrupt IC signaling, and the design of CARs against IC molecules. The current landscape indicates that the synergy of the fast-paced refinements of gene-editing technologies and synthetic biology and the increased comprehension of IC signaling will certainly translate into a novel and more effective immunotherapeutic approaches to treat patients with cancer.

Combination of genetically engineered T cells and immune checkpoint blockade for the treatment of cancer

Immune checkpoint (IC) blockade using monoclonal antibodies is currently one of the most successful immunotherapeutic interventions to treat cancer. By reinvigorating antitumor exhausted T cells, this approach can lead to durable clinical responses. However, the majority of patients either do not respond or present a short-lived response to IC blockade, in part due to a scarcity of tumor-specific T cells within the tumor microenvironment. Adoptive transfer of T cells genetically engineered to express chimeric antigen receptors (CARs) or engineered T-cell receptors (TCRs) provide the necessary tumor-specific immune cell population to target cancer cells. However, this therapy has been considerably ineffective against solid tumors in part due to IC-mediated immunosuppressive effects within the tumor microenvironment. These limitations could be overcome by associating adoptive cell transfer of genetically engineered T cells and IC blockade. In this comprehensive review, we highlight the strategies and outcomes of preclinical and clinical attempts to disrupt IC signaling in adoptive T-cell transfer against cancer. These strategies include combined administration of genetically engineered T cells and IC inhibitors, engineered T cells with intrinsic modifications to disrupt IC signaling, and the design of CARs against IC molecules. The current landscape indicates that the synergy of the fast-paced refinements of gene-editing technologies and synthetic biology and the increased comprehension of IC signaling will certainly translate into a novel and more effective immunotherapeutic approaches to treat patients with cancer.

Strategies to Enhance the Therapeutic Efficacy, Applicability, and Safety of Genetically Engineered Immune Cells.

The field of cell therapy is leading a paradigm shift in drug development. The recent convergence of several fields, including immunology, genetics, and synthetic biology, now allows for the introduction of artificial receptors and the design of entire genetic circuitries to finely program the behavior of injected cells. A prime example of these next-generation living drugs comes in the form of T cells expressing chimeric antigen receptors (CARs), which have already demonstrated definitive evidence of therapeutic efficacy against some hematological malignancies. However, several obstacles still restrict the antitumor efficacy of and impair the widespread use of CAR-T cells. Critical challenges include limited persistence and antitumor activity in vivo, antigen escape, scarcity of suitable single markers for targeting, and therapy-related toxicity. Nevertheless, intense research activity in this field has resulted in a plethora of creative solutions to address each of these limitations. In this review, we provide a comprehensive snapshot of the current strategies used to enhance the therapeutic efficacy, applicability, and safety of genetically engineered immune cells to treat cancer.

Human and mouse melanoma cells recapitulate an EMT-like program in response to mesenchymal stromal cells secretome.

The mechanisms underlying the propensity of melanomas to metastasize are not completely understood. We hypothesized that melanoma cells are capable of promptly activating an epithelial-to-mesenchymal transition (EMT)-like profile in response to stroma-derived factors. Thus, we investigated the role of mesenchymal stromal cells (MSCs), a cell population considered as a precursor of tumor stroma, on the activation of an EMT-like profile and acquisition of metastatic traits in melanoma cells. After subcutaneous co-injection with mouse B16 melanoma cells, MSCs occupied perivascular sites within tumors and enhanced B16 metastasis to the lungs. In vitro, MSCs' secretome activated an EMT-like profile in B16 cells, reducing their avidity to fibronectin, and increasing their motility and invasiveness. These effects were abrogated upon blocking of MET phosphorylation in B16 cells using small molecule inhibitors. MSCs also activated an EMT-like profile in human melanoma cells from different stages of progression. Activation of EMT in human cells was associated with increased levels of p-STAT1 and p-STAT3. In conclusion, both mouse and human melanoma cells are equipped to activate an EMT-like program and acquire metastatic traits through the activation of distinct pathways by MSCs' secretome.

Metabolic and proliferation evaluation of human adipose-derived mesenchymal stromal cells (ASC) in different culture medium volumes: standardization of static culture.

Adipose-derived mesenchymal stromal/stem cells (ASC) have acquired a prominent role in tissue engineering and regenerative medicine. However, the standardization of basic culture procedures in this cellular type is still not well established according to the main qualitative cellular attributes. We evaluate the cell growth profile of human ASC in a different culture medium volumes and their nutritional composition utilizing static cultivation. Culture medium volumes (5, 10 and 15 mL/25 cm2) in T-flasks were evaluated by kinetic parameters and the metabolic composition was determined by biochemical analysis and Fourier transform infrared (FT-IR) absorption spectroscopy. 50% renewal of culture medium volume every 48 h was adopted. Immunophenotypic characterization and cell differentiation were performed. There was no difference (p > 0.05) in the kinetic parameters of cell proliferation between the culture medium volumes or in FT-IR composition. However, the concentrations of glucose, glutamine, lactate, and glutamate varied significantly during the cultivation process as a function of the medium volume. ASC presented specific antigens and differentiation potential of mesenchymal stromal/stem cells. It was concluded that the minimal culture medium volume (5 mL/25 cm2 in static culture) was sufficient to maintain the stability, potency, and growth of ASC, representing an economic and safe standardization for this cell culture process.