Xuri W25 Cell Expansion System: Product Overview and Demo

The choice between in vivo and ex vivo cell expansion for CAR-T cell therapy has important implications. Ex vivo expansion allows for controlled manipulation and quality control before infusion. In vivo expansion minimizes laboratory manipulation, potentially preserving the natural cell phenotype. Research is comparing the immunophenotype and function of cells expanded in vivo versus those expanded ex vivo before infusion. The in vivo expansion approach may be particularly important for patients who are too debilitated for extensive laboratory manipulation. Epigenetic modulation using small molecule compounds can enhance CAR-T cell function. Research has shown that treating CAR-T cells with certain compounds for 3-5 days can improve their ability to kill target cells both in vitro and in vivo. This approach allows for phenotypic imprinting of cells during a short laboratory culture period.

Cell therapy uses cells for therapeutic purposes, with applications in cartilage repair (extracting, amplifying, and injecting cells) and myocardial infarction (using bone marrow stem cells to promote blood vessel formation). Cells are specialized units performing specific functions: neurons transmit impulses, melanocytes produce melanin, and beta cells produce insulin. The immune system includes B cells producing antibodies that neutralize pathogens or mark them for elimination, and T cells that destroy infected cells. Cancer cells evade immune detection through inefficient antigen presentation, immunosuppressive cell recruitment, and interference with T cell activation. CAR T cell therapy combines T cell cytotoxicity with antibody antigen specificity. The manufacturing process involves collecting patient blood, isolating T cells, introducing vectors for CAR receptor expression, expanding cells in vitro, and infusing them back to target cancer cells. The first successful case was Emily Whitehead in 2012, treated for acute lymphoblastic leukemia at age five. In Brazil, CAR T therapy was first used in 2019 for diffuse large B-cell lymphoma at USP Ribeirão Preto, with tumor elimination observed within one month.

Initial NK cell adoptive transfer used apheresis with T cell depletion yielding only 10-20 million/kg. The breakthrough came with feeder cell-based expansion using K562 cells transduced with 4-1BB ligand and membrane-bound IL-21. This enables 2,000-fold expansion in 2 weeks and 30,000+ fold in 3 weeks, delivering doses up to 1 billion/kg. Expanded NK cells maintain high granzyme/perforin levels and rapid cytotoxic function. Critically, these cells tolerate cryopreservation with >90% viability, enabling manufacturing ahead of patient availability and eliminating fresh product timing constraints.

The manufacturing of CAR-T cells follows a standardized ex vivo process: (1) Lymphocytes are isolated from patient blood using apheresis, which separates lymphocytes from other blood components by density; (2) Cells undergo purification using density gradients (Percoll) and magnetic bead separation with monoclonal antibodies; (3) T-cells are activated using anti-CD3/CD28/CD137 beads and cytokines (IL-2, IL-7, IL-15) for at least 72 hours; (4) Activated cells are transduced with replication-deficient retroviral vectors (gamma retrovirus or lentivirus); (5) CAR-T cells are expanded using the same beads and cytokines; (6) The product is formulated in DMSO-containing solution and frozen. The entire process takes 2-3 weeks. Before administration, patients receive pre-conditioning chemotherapy (cyclophosphamide and fludarabine) that depletes white blood cells and creates a niche for CAR-T cell expansion.

In vivo CAR-T cell therapy represents a revolutionary approach to cancer treatment that eliminates the need for complex ex vivo cell manufacturing by using mRNA-LNP (lipid nanoparticle) platforms to deliver genetic information directly into the patient's body, enabling T-cells to produce CAR proteins internally and significantly reducing treatment costs and time compared to traditional methods.

Three-dimensional cell culture systems are classified into static (non-moving) and dynamic (irrigated) systems. Static systems include hanging drop cultures, removable floating devices, printing devices, galactose-related substrates, 3D scaffolds, and familiar dishes. Dynamic systems include spinner flasks, March automatic devices, marked reactors, and rotating bioreactors. Rotating bioreactors use clinostat principles from the 1920s, reducing gravitational pull on cells to overcome phototropism and geotropism. These systems allow cells to find themselves in more natural geometrical shapes, forming in vivo-like tissue structures that provide clinically relevant data.

Static systems and dynamic systems require different approaches. Static systems involve fixed arrangements, while dynamic systems involve moving components. The dynamic system can mix gases and create different conditions. The goal is to teach how to use gases in dynamic reactors to see the beauty of the technology.

Dynamic flow bioreactors provide significant advantages over static culture plates. In static cultures without media changes, cells show yellowing due to nutrient depletion and accumulate waste products. Daily media changes improve cell density but still result in gradual depletion. Semi-continuous dynamic flow systems continuously remove waste products while replenishing oxygen and nutrients, enabling significantly higher cell densities and better overall cell health compared to batch or static culture approaches.

T-cell expansion uses bioreactor systems with rocking motion to facilitate gas exchange. Unlike static cultures, rocking motion allows much more efficient gas exchange and higher cell concentrations. The system includes a filter that keeps cells contained while fresh media is perfused in and waste media removed, enabling scalable and controlled cell culture conditions.

Static culture systems include dome culture (embedding cells in extracellular matrix or hydrogel) and ultra-low attachment environments (specialized plates with anti-adherence coating that cause cells to aggregate). Hanging drop involves suspending cells in small droplets where gravity causes aggregation. Dynamic systems include orbital shakers (plates on planar shakers with gentle swirling) and bioreactor systems (impeller or stir-driven) for large-scale production.

This extensive section covers the complete physiology of tissue perfusion and its clinical markers. Tissue perfusion is defined as the continuous physiological process of blood volume distribution per unit of time and tissue weight to guarantee energy requirements. The three key concepts are: DO2 (oxygen delivery), VO2 (oxygen consumption), and oxygen extraction rate (normally 25-35%). The section covers the historical development of shock and lactate as markers, the physiological relationships governing oxygen transport, and the calculation of oxygen delivery using cardiac output and arterial oxygen content. The hemoglobin-oxygen dissociation curve is explained, including P50 (26-27 mmHg) and factors affecting curve shifts. Microcirculation (vessels <100 micrometers) is introduced as critical for gas exchange. Cellular metabolism is covered, explaining glycolysis, the Krebs cycle, and oxidative phosphorylation, with oxygen being essential specifically for Complex IV of oxidative phosphorylation. The section then transitions to clinical markers: ScvO2 (normal lower limit 65%), lactate (normal ≤2 mmol/L, mortality increases exponentially above 4 mmol/L), and the critical point in tissue perfusion where compensatory mechanisms fail.

To determine whether water in a living cell is cancerous or healthy, only three measurements are needed: electrical resistance, pH (acidity), and redox potential. These three parameters immediately indicate the health status of the cell's water.

The fundamental meaning of pH is that it determines whether oxygen will enter cells. When blood pH is alkaline (high pH), oxygen cannot enter cells effectively. When blood pH is normal, oxygen enters cells properly. This is the core mechanism by which pH affects cellular oxygenation and tissue oxygenation.

STS database analysis of 834 cardiac surgery patients demonstrated DO2I <280 mL/min/m² independently predicted increased morbidity, mortality, prolonged ventilation, and acute kidney injury within 72 hours. Hypothermia shifts the oxyhemoglobin dissociation curve leftward, increasing oxygen affinity and reducing tissue release—each degree Celsius decreases P50 by ~0.5 mmHg. Stored blood further impairs oxygen release through reduced 2,3-DPG levels. Two oxygenation strategies exist: alpha-stat maintains pH 7.4 at 37°C (adult preference); pH-stat maintains actual pH 7.4 at current temperature using CO2-O2 mixtures (pediatric preference). Normal physiology features a delivery-dependent zone where consumption increases with delivery up to ~400 mL/min/m², then plateaus. Disease states extend this zone abnormally, with consumption continuing to rise with higher delivery—an indicator of cellular dysfunction.

This comprehensive section covers fundamental cellular biology and physiological parameters: oxygen enters cells through diffusion; phagocytosis is cell eating; active transport requires energy; protein synthesis occurs in ribosomes; cell membrane contains cholesterol, phospholipids, and proteins but not galactose; cells obtain energy from carbohydrates; potassium has highest intracellular concentration; cellular respiration occurs in mitochondria; human cells contain 46 chromosomes in 23 pairs; sodium has highest extracellular concentration; normal body fluid pH is 7.35-7.45; saliva pH is 6.7-7.4; sweat pH is 4.3-4.7; milk pH is 6.5-6.7; blood pH is 7.35-7.45; urine pH is 4.5-8.0; gastric juice pH is 1.5-3.5; fibrous connective tissue includes tendons and ligaments; fluid connective tissue is blood; hard connective tissue is bone; ciliated epithelium is in respiratory tract; adipose tissue contains fatty substances; keratinized stratified epithelium is in skin, hair, and nails; albumin is most abundant plasma protein; fibrinogen is synthesized by liver; thrombocytopenia causes bleeding; zona fasciculata produces glucocorticoids; platelets are not leukocytes; kidney-shaped nucleus is in monocytes; T cell maturation occurs in thymus; blood circulation discovered by William Harvey; normal erythrocyte count in males is 10-20 million per cubic millimeter; microcytic anemia is caused by iron deficiency.