Three-Dimensional Human Bone Marrow Organoids for the Study and Application of Normal and Abnormal Hematoimmunopoiesis.

Hematoimmunopoiesis takes place in the adult human bone marrow (BM), which is composed of heterogeneous niches with complex architecture that enables tight regulation of homeostatic and stress responses. There is a paucity of representative culture systems that recapitulate the heterogeneous three-dimensional (3D) human BM microenvironment and that can endogenously produce soluble factors and extracellular matrix that deliver culture fidelity for the study of both normal and abnormal hematopoiesis. Native BM lymphoid populations are also poorly represented in current in vitro and in vivo models, creating challenges for the study and treatment of BM immunopathology. BM organoid models leverage normal 3D organ structure to recreate functional niche microenvironments. Our focus herein is to review the current state of the art in the use of 3D BM organoids, focusing on their capacities to recreate critical quality attributes of the in vivo BM microenvironment for the study of human normal and abnormal hematopoiesis.

Are all cytokine storms the same?

Cytokine release syndrome (CRS) is the result of massive pro-inflammatory cytokine release and imbalance in the absence of adequate immunomodulation from signals such as interleukin (IL)-10, resulting in ongoing inflammation, tissue damage and death if left uncontrolled. Although CRS can result from different pro-inflammatory insults, the treatments proposed are similar, regardless of the phase of response. SARS-CoV-2 causes COVID-19, and CRS has been a defining feature of severe disease. Common approaches to treating CRS in other conditions are now applied to COVID-19 and, although some patients respond, it begs the following questions: (1) are all cytokine storms the same regardless of initiating insult, (2) can treatments be considered equally for all CRS events at any phase of the response, (3) can CRS be predicted based on dynamic acute biomarkers and, (4) should patients with CRS undergo long-term monitoring for secondary effects? The aim of this commentary is not to provide a review of COVID-19 pathophysiology or of cytokine storm, but rather to establish a foundation which could act as a platform to inform treatment approaches to CRS, regardless of cause, and the short- and long-term follow-up which may be necessary for affected patients.

Myelopoiesis of acute inflammation: lessons from TGN1412-induced cytokine storm.

TGN1412, a superagonist monoclonal antibody targeting CD28, caused cytokine storm in six healthy volunteers in a first-in-man study in 2006. Despite clinical improvement and termination of the cytokine release syndrome within days, anemia persisted in all patients with hemoglobin reaching baseline levels as much as 6 months later. Granulocytic dysplasia continued for 20 days in association with increased expression of CD69 and IL-4, but reduced IL-10; with resolution, this profile reversed to higher IL-10 expression and counter-balanced circannual cycling of IL-4 and IL-10 thereafter over 7 months. Along with immune cell subset and cytokine correlates monitored over 2 years, these observations offer unique insights into the expected changes in myelopoiesis and natural resolution in otherwise healthy young individuals in response to acute inflammation and cytokine storm in the absence of concomitant infection or comorbidity.

Patients with gastrointestinal irritability after TGN1412-induced cytokine storm displayed selective expansion of gut-homing αß and γδT cells.

Following infusion of the anti-CD28 superagonist monoclonal antibody TGN1412, three of six previously healthy, young male recipients developed gastrointestinal irritability associated with increased expression of 'gut-homing' integrin ß7 on peripheral blood αßT cells. This subset of patients with intestinal symptoms also displayed a striking and persistent expansion of putative Vδ2+ γδT cells in the circulation which declined over a 2-year period following drug infusion, concordant with subsiding gut symptoms. These data demonstrate that TGN1412-induced gastrointestinal symptoms were associated with dysregulation of the 'gut-homing' pool of blood αß and γδT cells, induced directly by the antibody and/or arising from the subsequent cytokine storm.

Immune reconstitution and clinical recovery following anti-CD28 antibody (TGN1412)-induced cytokine storm.

Cytokine storm can result from cancer immunotherapy or certain infections, including COVID-19. Though short-term immune-related adverse events are routinely described, longer-term immune consequences and sequential immune monitoring are not as well defined. In 2006, six healthy volunteers received TGN1412, a CD28 superagonist antibody, in a first-in-man clinical trial and suffered from cytokine storm. After the initial cytokine release, antibody effect-specific immune monitoring started on Day + 10 and consisted mainly of evaluation of dendritic cell and T-cell subsets and 15 serum cytokines at 21 time-points over 2 years. All patients developed problems with concentration and memory; three patients were diagnosed with mild-to-moderate depression. Mild neutropenia and autoantibody production was observed intermittently. One patient suffered from peripheral dry gangrene, required amputations, and had persistent Raynaud's phenomenon. Gastrointestinal irritability was noted in three patients and coincided with elevated γδT-cells. One had pruritus associated with elevated IgE levels, also found in three other asymptomatic patients. Dendritic cells, initially undetectable, rose to normal within a month. Naïve CD8+ T-cells were maintained at high levels, whereas naïve CD4+ and memory CD4+ and CD8+ T-cells started high but declined over 2 years. T-regulatory cells cycled circannually and were normal in number. Cytokine dysregulation was especially noted in one patient with systemic symptoms. Over a 2-year follow-up, cognitive deficits were observed in all patients following TGN1412 infusion. Some also had signs or symptoms of psychological, mucosal or immune dysregulation. These observations may discern immunopathology, treatment targets, and long-term monitoring strategies for other patients undergoing immunotherapy or with cytokine storm.

Ruxolitinib vs best available therapy for ET intolerant or resistant to hydroxycarbamide.

Treatments for high-risk essential thrombocythemia (ET) address thrombocytosis, disease-related symptoms, as well as risks of thrombosis, hemorrhage, transformation to myelofibrosis, and leukemia. Patients resistant/intolerant to hydroxycarbamide (HC) have a poor outlook. MAJIC (ISRCTN61925716) is a randomized phase 2 trial of ruxolitinib (JAK1/2 inhibitor) vs best available therapy (BAT) in ET and polycythemia vera patients resistant or intolerant to HC. Here, findings of MAJIC-ET are reported, where the modified intention-to-treat population included 58 and 52 patients randomized to receive ruxolitinib or BAT, respectively. There was no evidence of improvement in complete response within 1 year reported in 27 (46.6%) patients treated with ruxolitinib vs 23 (44.2%) with BAT (P = .40). At 2 years, rates of thrombosis, hemorrhage, and transformation were not significantly different; however, some disease-related symptoms improved in patients receiving ruxolitinib relative to BAT. Molecular responses were uncommon; there were 2 complete molecular responses (CMR) and 1 partial molecular response in CALR-positive ruxolitinib-treated patients. Transformation to myelofibrosis occurred in 1 CMR patient, presumably because of the emergence of a different clone, raising questions about the relevance of CMR in ET patients. Grade 3 and 4 anemia occurred in 19% and 0% of ruxolitinib vs 0% (both grades) in the BAT arm, and grade 3 and 4 thrombocytopenia in 5.2% and 1.7% of ruxolitinib vs 0% (both grades) of BAT-treated patients. Rates of discontinuation or treatment switching did not differ between the 2 trial arms. The MAJIC-ET trial suggests that ruxolitinib is not superior to current second-line treatments for ET. This trial was registered at www.isrctn.com as #ISRCTN61925716.

Energy-based culture medium design for biomanufacturing optimization: A case study in monoclonal antibody production by GS-NS0 cells.

Demand for high-value biologics, a rapidly growing pipeline, and pressure from competition, time-to-market and regulators, necessitate novel biomanufacturing approaches, including Quality by Design (QbD) principles and Process Analytical Technologies (PAT), to facilitate accelerated, efficient and effective process development platforms that ensure consistent product quality and reduced lot-to-lot variability. Herein, QbD and PAT principles were incorporated within an innovative in vitro-in silico integrated framework for upstream process development (UPD). The central component of the UPD framework is a mathematical model that predicts dynamic nutrient uptake and average intracellular ATP content, based on biochemical reaction networks, to quantify and characterize energy metabolism and its adaptive response, metabolic shifts, to maintain ATP homeostasis. The accuracy and flexibility of the model depends on critical cell type/product/clone-specific parameters, which are experimentally estimated. The integrated in vitro-in silico platform and the model's predictive capacity reduced burden, time and expense of experimentation resulting in optimal medium design compared to commercially available culture media (80% amino acid reduction) and a fed-batch feeding strategy that increased productivity by 129%. The framework represents a flexible and efficient tool that transforms, improves and accelerates conventional process development in biomanufacturing with wide applications, including stem cell-based therapies.

Vosaroxin and vosaroxin plus low-dose Ara-C (LDAC) vs low-dose Ara-C alone in older patients with acute myeloid leukemia.

The development of new treatments for older patients with acute myeloid leukemia is an active area, but has met with limited success. Vosaroxin, a quinolone-derived intercalating agent has several properties that could prove beneficial. Initial clinical studies showed it to be well-tolerated in older patients with relapsed/refractory disease. In vitro data suggested synergy with cytarabine (Ara-C). To evaluate vosaroxin, we performed 2 randomized comparisons within the "Pick a Winner" program. A total of 104 patients were randomized to vosaroxin vs low-dose Ara-C (LDAC) and 104 to vosaroxin + LDAC vs LDAC. When comparing vosaroxin with LDAC, neither response rate (complete recovery [CR]/complete recovery with incomplete count recovery [CRi], 26% vs 30%; odds ratio [OR], 1.16 (0.49-2.72); P = .7) nor 12-month survival (12% vs 31%; hazard ratio [HR], 1.94 [1.26-3.00]; P = .003) showed benefit for vosaroxin. Likewise, in the vosaroxin + LDAC vs LDAC comparison, neither response rate (CR/CRi, 38% vs 34%; OR, 0.83 [0.37-1.84]; P = .6) nor survival (33% vs 37%; HR, 1.30 [0.81-2.07]; P = .3) was improved. A major reason for this lack of benefit was excess early mortality in the vosaroxin + LDAC arm, most obviously in the second month following randomization. At its first interim analysis, the Data Monitoring and Ethics Committee recommended closure of the vosaroxin-containing trial arms because a clinically relevant benefit was unlikely.

Towards unravelling the kinetics of an acute myeloid leukaemia model system under oxidative and starvation stress: a comparison between two- and three-dimensional cultures.

A great challenge when conducting ex vivo studies of leukaemia is the construction of an appropriate experimental platform that would recapitulate the bone marrow (BM) environment. Such a 3D scaffold system has been previously developed in our group [1]. Additionally to the BM architectural characteristics, parameters such as oxygen and glucose concentration are crucial as their value could differ between patients as well as within the same patient at different stages of treatment, consequently affecting the resistance of leukaemia to chemotherapy. The effect of oxidative and glucose stress-at levels close to human physiologic ones-on the proliferation and metabolic evolution of an AML model system (K-562 cell line) in conventional 2D cultures as well as in 3D scaffolds were studied. We observed that the K-562 cell line can proliferate and remain alive for 2 weeks in medium with glucose close to physiological levels both in 20 and 5% O2. We report interesting differences on the cellular response to the environmental, i.e., oxidative and/or nutritional stress stimuli in 2D and 3D. Higher adaptation to oxidative stress under non-starving conditions is observed in the 3D system. The glucose level in the medium has more impact on the cellular proliferation in the 3D compared to the 2D system. These differences can be of significant importance both when applying chemotherapy in vitro and also when constructing mathematical tools for optimisation of disease treatment.

A Spatiotemporal Microenvironment Model to Improve Design of a Three-Dimensional Bioreactor for Red Cell Production.

Cellular microenvironments provide stimuli, including paracrine and autocrine growth factors and physicochemical cues, which support efficient in vivo cell production unmatched by current in vitro biomanufacturing platforms. While three-dimensional (3D) culture systems aim to recapitulate niche architecture and function of the target tissue/organ, they are limited in accessing spatiotemporal information to evaluate and optimize in situ cell/tissue process development. Herein, a mathematical modeling framework is parameterized by single-cell phenotypic imaging and multiplexed biochemical assays to simulate the nonuniform tissue distribution of nutrients/metabolites and growth factors in cell niche environments. This model is applied to a bone marrow mimicry 3D perfusion bioreactor containing dense stromal and hematopoietic tissue with limited red blood cell (RBC) egress. The model characterized an imbalance between endogenous cytokine production and nutrient starvation within the microenvironmental niches and recommended increased cell inoculum density and enhanced medium exchange, guiding the development of a miniaturized prototype bioreactor. The second-generation prototype improved the distribution of nutrients and growth factors and supported a 50-fold increase in RBC production efficiency. This image-informed bioprocess modeling framework leverages spatiotemporal niche information to enhance biochemical factor utilization and improve cell manufacturing in 3D systems. Impact statement Three-dimensional (3D) culture systems are becoming increasingly important because they recapitulate the architecture and, consequently, physiological function of the target tissue/organ. Design and optimization of these 3D biomanufacturing platforms require evaluation of in situ spatiotemporal information. We have developed an integrated experimental-computational framework that captures the spatiotemporal distribution of cells, nutrients, and cytokines within a marrow biomimicry perfusion bioreactor. The model simulated biochemical factor utilization and guided the design of an improved second-generation bioreactor that achieved 50-fold increase in RBC production with improved cost efficiency. Such a modeling framework provides an essential platform for the optimization of 3D biomanufacturing systems.

Clinical outcomes in patients with relapsed/refractory FLT3-mutated acute myeloid leukemia treated with gilteritinib who received prior midostaurin or sorafenib.

The fms-like tyrosine kinase 3 (FLT3) inhibitor gilteritinib is indicated for relapsed or refractory (R/R) FLT3-mutated acute myeloid leukemia (AML), based on its observed superior response and survival outcomes compared with salvage chemotherapy (SC). Frontline use of FLT3 tyrosine kinase inhibitors (TKIs) midostaurin and sorafenib may contribute to cross-resistance to single-agent gilteritinib in the R/R AML setting but has not been well characterized. To clarify the potential clinical impact of prior TKI use, we retrospectively compared clinical outcomes in patients with R/R FLT3-mutated AML in the CHRYSALIS and ADMIRAL trials who received prior midostaurin or sorafenib against those without prior FLT3 TKI exposure. Similarly high rates of composite complete remission (CRc) were observed in patients who received a FLT3 TKI before gilteritinib (CHRYSALIS, 42%; ADMIRAL, 52%) and those without prior FLT3 TKI therapy (CHRYSALIS, 43%; ADMIRAL, 55%). Among patients who received a prior FLT3 TKI in ADMIRAL, a higher CRc rate (52%) and trend toward longer median overall survival was observed in the gilteritinib arm versus the SC arm (CRc = 20%; overall survival, 5.1 months; HR = 0.602; 95% CI: 0.299, 1.210). Remission duration was shorter with prior FLT3 TKI exposure. These findings support gilteritinib for FLT3-mutated R/R AML after prior sorafenib or midostaurin.

Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412.

Six healthy young male volunteers at a contract research organization were enrolled in the first phase 1 clinical trial of TGN1412, a novel superagonist anti-CD28 monoclonal antibody that directly stimulates T cells. Within 90 minutes after receiving a single intravenous dose of the drug, all six volunteers had a systemic inflammatory response characterized by a rapid induction of proinflammatory cytokines and accompanied by headache, myalgias, nausea, diarrhea, erythema, vasodilatation, and hypotension. Within 12 to 16 hours after infusion, they became critically ill, with pulmonary infiltrates and lung injury, renal failure, and disseminated intravascular coagulation. Severe and unexpected depletion of lymphocytes and monocytes occurred within 24 hours after infusion. All six patients were transferred to the care of the authors at an intensive care unit at a public hospital, where they received intensive cardiopulmonary support (including dialysis), high-dose methylprednisolone, and an anti-interleukin-2 receptor antagonist antibody. Prolonged cardiovascular shock and acute respiratory distress syndrome developed in two patients, who required intensive organ support for 8 and 16 days. Despite evidence of the multiple cytokine-release syndrome, all six patients survived. Documentation of the clinical course occurring over the 30 days after infusion offers insight into the systemic inflammatory response syndrome in the absence of contaminating pathogens, endotoxin, or underlying disease.

Stem cell bioprocessing: fundamentals and principles.

In recent years, the potential of stem cell research for tissue engineering-based therapies and regenerative medicine clinical applications has become well established. In 2006, Chung pioneered the first entire organ transplant using adult stem cells and a scaffold for clinical evaluation. With this a new milestone was achieved, with seven patients with myelomeningocele receiving stem cell-derived bladder transplants resulting in substantial improvements in their quality of life. While a bladder is a relatively simple organ, the breakthrough highlights the incredible benefits that can be gained from the cross-disciplinary nature of tissue engineering and regenerative medicine (TERM) that encompasses stem cell research and stem cell bioprocessing. Unquestionably, the development of bioprocess technologies for the transfer of the current laboratory-based practice of stem cell tissue culture to the clinic as therapeutics necessitates the application of engineering principles and practices to achieve control, reproducibility, automation, validation and safety of the process and the product. The successful translation will require contributions from fundamental research (from developmental biology to the 'omics' technologies and advances in immunology) and from existing industrial practice (biologics), especially on automation, quality assurance and regulation. The timely development, integration and execution of various components will be critical-failures of the past (such as in the commercialization of skin equivalents) on marketing, pricing, production and advertising should not be repeated. This review aims to address the principles required for successful stem cell bioprocessing so that they can be applied deftly to clinical applications.

Dynamic human erythropoiesis in a three-dimensional perfusion bone marrow biomimicry.

Traditional culture systems for human erythropoiesis lack microenvironmental niches, spatial marrow gradients and dense cellularity rendering them incapable of effectively translating marrow physiology ex vivo. Herein, a bio-inspired three-dimensional (3D) perfusion bioreactor was engineered and inoculated with unselected single donor umbilical cord blood mononuclear cells (CBMNCs). Functional stromal and hematopoietic environments supporting long-term erythropoiesis were generated using defined medium supplemented only with stem cell factor (SCF) and erythropoietin (EPO) at near physiological concentrations. Quantitative 3D image analyses spatiotemporally mapped 21 multi-lineal cell distributions and interactions within multiple microenvironments that secreted extracellular matrix proteins and at least 16 endogenous hematopoietic and stromal growth factors. Tissue-like culture densities (≥2∙109 cells/mL), 1000-fold above flask cultures, were attained with continuous erythropoiesis and erythrocyte harvest. We propose this physiologically-relevant system for understanding normal and abnormal erythropoiesis, as well as for drug testing and/or discovery aimed at clinical translation.

RGD-functionalized polyurethane scaffolds promote umbilical cord blood mesenchymal stem cell expansion and osteogenic differentiation.

Biomimetic materials are essential for the production of clinically relevant bone grafts for bone tissue engineering applications. Their ability to modulate stem cell proliferation and differentiation can be used to harness the regenerative potential of those cells and optimize the efficiency of engineered bone grafts. The arginyl-glycyl-aspartic acid (RGD) peptide has been recognized as the adhesion motif of various extracellular matrix proteins and can affect stem cell behaviour in biomaterials. Attempts to functionalize biomaterials with RGD have been limited to a maximum of 1- to 3-mm thickness scaffolds, overlooking the issue of core infiltration that represents a major hurdle in developing real thickness scaffolds. Herein, we present the cross-linking of RGD on the surface of "real thickness" (5 × 5 × 5 mm) porous polyurethane scaffolds (PU-RGD), to be used for the expansion and osteogenic differentiation of umbilical cord blood mesenchymal stem cells (UCB MSCs). RGD-functionalized scaffolds increased initial cell adhesion (1.5-fold to twofold) and achieved a 3.4-fold increase in cell numbers at the end of culture compared with a 1.5-fold increase in non-functionalized controls. Homogenous cell infiltration to the scaffold core was observed in the PU-RGD scaffolds. Importantly, PU-RGD scaffolds were able to enhance the osteogenic differentiation of UCB MSCs. Osteogenic gene and protein expression increased in scaffolds functionalized with 100 µg/ml RGD. Higher RGD concentrations (200 µg/ml) were less efficient in stimulating osteogenic differentiation. We conclude that robust RGD tethering to 3D PU "real thickness" scaffolds is possible and that it promotes core infiltration, expansion, and osteogenic differentiation of UCB MSCs for the purposes of bone regeneration.

Oxidized alginate hydrogels with the GHK peptide enhance cord blood mesenchymal stem cell osteogenesis: A paradigm for metabolomics-based evaluation of biomaterial design.

Oxidized alginate hydrogels are appealing alternatives to natural alginate due to their favourable biodegradability profiles and capacity to self-crosslink with amine containing molecules facilitating functionalization with extracellular matrix cues, which enable modulation of stem cell fate, achieve highly viable 3-D cultures, and promote cell growth. Stem cell metabolism is at the core of cellular fate (proliferation, differentiation, death) and metabolomics provides global metabolic signatures representative of cellular status, being able to accurately identify the quality of stem cell differentiation. Herein, umbilical cord blood mesenchymal stem cells (UCB MSCs) were encapsulated in novel oxidized alginate hydrogels functionalized with the glycine-histidine-lysine (GHK) peptide and differentiated towards the osteoblastic lineage. The ADA-GHK hydrogels significantly improved osteogenic differentiation compared to gelatin-containing control hydrogels, as demonstrated by gene expression, alkaline phosphatase activity and bone extracellular matrix deposition. Metabolomics revealed the high degree of metabolic heterogeneity in the gelatin-containing control hydrogels, captured the enhanced osteogenic differentiation in the ADA-GHK hydrogels, confirmed the similar metabolism between differentiated cells and primary osteoblasts, and elucidated the metabolic mechanism responsible for the function of GHK. Our results suggest a novel paradigm for metabolomics-guided biomaterial design and robust stem cell bioprocessing. STATEMENT OF SIGNIFICANCE: Producing high quality engineered bone grafts is important for the treatment of critical sized bone defects. Robust and sensitive techniques are required for quality assessment of tissue-engineered constructs, which result to the selection of optimal biomaterials for bone graft development. Herein, we present a new use of metabolomics signatures in guiding the development of novel oxidised alginate-based hydrogels with umbilical cord blood mesenchymal stem cells and the glycine-histidine-lysine peptide, demonstrating that GHK induces stem cell osteogenic differentiation. Metabolomics signatures captured the enhanced osteogenesis in GHK hydrogels, confirmed the metabolic similarity between differentiated cells and primary osteoblasts, and elucidated the metabolic mechanism responsible for the function of GHK. In conclusion, our results suggest a new paradigm of metabolomics-driven design of biomaterials.

Stem cell biomanufacturing under uncertainty: A case study in optimizing red blood cell production.

As breakthrough cellular therapy discoveries are translated into reliable, commercializable applications, effective stem cell biomanufacturing requires systematically developing and optimizing bioprocess design and operation. This article proposes a rigorous computational framework for stem cell biomanufacturing under uncertainty. Our mathematical tool kit incorporates: high-fidelity modeling, single variate and multivariate sensitivity analysis, global topological superstructure optimization, and robust optimization. The advantages of the proposed bioprocess optimization framework using, as a case study, a dual hollow fiber bioreactor producing red blood cells from progenitor cells were quantitatively demonstrated. The optimization phase reduces the cost by a factor of 4, and the price of insuring process performance against uncertainty is approximately 15% over the nominal optimal solution. Mathematical modeling and optimization can guide decision making; the possible commercial impact of this cellular therapy using the disruptive technology paradigm was quantitatively evaluated.

Ceramic Hollow Fibre Constructs for Continuous Perfusion and Cell Harvest from 3D Hematopoietic Organoids.

Tissue vasculature efficiently distributes nutrients, removes metabolites, and possesses selective cellular permeability for tissue growth and function. Engineered tissue models have been limited by small volumes, low cell densities, and invasive cell extraction due to ineffective nutrient diffusion and cell-biomaterial attachment. Herein, we describe the fabrication and testing of ceramic hollow fibre membranes (HFs) able to separate red blood cells (RBCs) and mononuclear cells (MNCs) and be incorporated into 3D tissue models to improve nutrient and metabolite exchange. These HFs filtered RBCs from human umbilical cord blood (CB) suspensions of 20% RBCs to produce 90% RBC filtrate suspensions. When incorporated within 5 mL of 3D collagen-coated polyurethane porous scaffold, medium-perfused HFs maintained nontoxic glucose, lactate, pH levels, and higher cell densities over 21 days of culture in comparison to nonperfused 0.125 mL scaffolds. This hollow fibre bioreactor (HFBR) required a smaller per-cell medium requirement and operated at cell densities > 10-fold higher than current 2D methods whilst allowing for continuous cell harvest through HFs. Herein, we propose HFs to improve 3D cell culture nutrient and metabolite diffusion, increase culture volume and cell density, and continuously harvest products for translational cell therapy biomanufacturing protocols.

Intelligent bioprocessing for haemotopoietic cell cultures using monitoring and design of experiments.

The need for successful ex-vivo expansion and directed differentiation of haematopoietic stem cells (HSCs) for therapeutic applications has increased over the past decade. Haematopoietic cell cultures are complex and full characterisation of the process environment has yet to be achieved. The complexity and transient nature of HSC cultures make the identification, maintenance and control of optimal operating conditions challenging. Application of real-time, on-line monitoring techniques and process control strategies enhances the ability to operate bioprocesses of desired reproducibility and high product quality. In this review, we discussed the methods by which in vitro culture information necessary for bioprocess control may be obtained, including process considerations, monitoring and analytical tools, and design of experiments (DOE). The successful application of these tools may result in time- and cost-effective cultures for directed differentiation and expansion of haematopoietic components intended for clinical use.

Human embryonic and induced pluripotent stem cells maintain phenotype but alter their metabolism after exposure to ROCK inhibitor.

Human pluripotent stem cells (hPSCs) are adhesion-dependent cells that require cultivation in colonies to maintain growth and pluripotency. Robust differentiation protocols necessitate single cell cultures that are achieved by use of ROCK (Rho kinase) inhibitors. ROCK inhibition enables maintenance of stem cell phenotype; its effects on metabolism are unknown. hPSCs were exposed to 10 µM ROCK inhibitor for varying exposure times. Pluripotency (TRA-1-81, SSEA3, OCT4, NANOG, SOX2) remained unaffected, until after prolonged exposure (96 hrs). Gas chromatography-mass spectrometry metabolomics analysis identified differences between ROCK-treated and untreated cells as early as 12 hrs. Exposure for 48 hours resulted in reduction in glycolysis, glutaminolysis, the citric acid (TCA) cycle as well as the amino acids pools, suggesting the adaptation of the cells to the new culture conditions, which was also reflected by the expression of the metabolic regulators, mTORC1 and tp53 and correlated with cellular proliferation status. While gene expression and protein levels did not reveal any changes in the physiology of the cells, metabolomics revealed the fluctuating state of the metabolism. The above highlight the usefulness of metabolomics in providing accurate and sensitive information on cellular physiological status, which could lead to the development of robust and optimal stem cell bioprocesses.