Expansion of human induced pluripotent stem cells (hiPSCs) in 3D bioreactors for extracorporeal liver support

Acute liver failure (ALF) is a life-threatening condition, which to date can only be treated by supportive care or liver transplantation. The use of hiPSC-derived hepatocyte-like cells (HLCs) offers a promising therapeutic alternative which, however, requires sufficient hiPSC quantities at high cell quality and purity. Therefore, the aim of the first part of this thesis was to investigate the expansion of hiPSCs in perfusion-based, 3D hollow-fiber bioreactors combined with non-invasive online monitoring of oxygen for culture surveillance. The first study examines the effect of the initial cell density on the quantitative and qualitative expansion outcome. Analysis of the expansion rates, which were determined by cell counting and performing the CellTiter-Blueâ Assay, revealed a more than 100-fold expansion at low initial cell densities (2,9 - 3,3 x 106 cells/mL) compared to a 28-fold expansion at higher initial cell densities (16,6 x 106 cells/mL). Furthermore, a higher rate of spontaneous cell differentiation occurred in bioreactors inoculated with higher cell densities. To conclude, lower initial cell densities in the range of 3 x 106 cells/mL lead to better quantitative and qualitative expansion results compared to higher initial cell densities. In the second study, the use of continuous measurement of oxygen for online culture control was evaluated. Calculated oxygen uptake rates (OURs), glucose consumption rates (GCRs) and lactate production rates (LPRs) revealed a highly significant correlation (p < 0.0001), indicating that oxygen is equivalent to glucose as parameter for hiPSC expansion while providing an accurate real-time monitoring of the hiPSC culture development. As a result of both studies, the conditions for an optimized expansion of hiPSCs in 3D bioreactors under continuous online surveillance of oxygen were successfully established. Subject of the second part of this thesis were basic studies on the co-cultivation of hiPSCs with human umbilical vein endothelial cells (HUVECs) for an improvement of the hepatic differentiation of hiPSCs. The study was aimed at determining a medium composition supporting both, the hepatic differentiation of hiPSCs as well as the maintenance of co-cultured HUVECs. The results revealed that a mixture of hepatocyte culture medium (HCM) and endothelial growth medium (EGM) at a 1:1 ratio, as well as a mixture of HCM and EGM supplements (without the base medium) supported the maintenance of HUVEC cultures. Thus, suitable medium compositions for following experiments on co-cultures of hiPSCs and HUVECs were successfully identified.Akutes Leberversagen führt zu einem lebensbedrohlichen klinischen Zustand; therapeutische Möglichkeiten sind derzeit limitiert auf unterstützende Verfahren und die Lebertransplantation als Ultima Ratio. Die Verwendung von aus humanen induzierten pluripotenten Stammzellen (hiPSCs) gebildeten Hepatozyten, sog. hepatocyte-like cells (HLCs), stellt eine vielversprechende therapeutische Alternative dar, für die jedoch ausreichende Zellmengen in hoher Qualität und Reinheit benötigt werden. Ziel des ersten Teils dieser Arbeit war es, die Expansion von hiPSCs in perfusionsbasierten 3DHohlfaser-Bioreaktoren in Kombination mit einem nicht-invasiven Online-Monitoring-Verfahren von Sauerstoff zur Kulturüberwachung zu untersuchen. Die erste Studie beschäftigt sich mit dem Einfluss der initialen Zelldichte auf das quantitative sowie qualitative Expansionsergebnis. Die Analyse der Expansionsraten, ermittelt durch Zellzählung und den CellTiter-Blueâ Assay, ergab eine mehr als 100-fache Expansion in Bioreaktoren mit einer geringeren initialen Zelldichte (2,9 - 3,3 x 106 Zellen/mL). Bioreaktoren, die mit einer höheren Zellzahl befüllt wurden (16,6 x 106 Zellen/mL), zeigten hingegen nur eine 28-fache Expansion. Außerdem wurde anhand von Genexpressionsanalysen und immunhistologischen Untersuchungen in diesen Bioreaktoren auch eine höhere Rate einer spontanen, ungezielten Differenzierung festgestellt. Aus den Ergebnissen lässt sich schließen, dass eine eher geringe Zelldichte im Bereich von 3 x 106 Zellen/mL sowohl quantitativ als auch qualitativ zu besseren Expansionsergebnissen als eine höhere Zelldichte führt. In der zweiten Studie wurde die Verwendung einer kontinuierlichen Sauerstoffmessung zur engmaschigen Überwachung des Expansionsprozesses untersucht. Die errechneten Sauerstoffaufnahmeraten (OURs), Glukoseverbrauchsraten (GCRs) und Laktatproduktionsraten (LPRs) zeigten eine hoch signifikante Korrelation (p < 0.0001) und wiesen somit darauf hin, dass Sauerstoff in gleichem Maße wie Glukose zum Monitoring der hiPSC-Expansion in dem untersuchten Bioreaktorsystem geeignet ist, jedoch mit dem zusätzlichen Vorteil, die Kulturentwicklung in Echtzeit darzustellen. Mit diesen beiden Studien wurden erfolgreich die Bedingungen für eine optimierte Expansion von hiPSCs in 3D-Bioreaktoren unter kontinuierlicher Online-Sauerstoffüberwachung etabliert. Inhalt des zweiten Teils dieser Arbeit waren grundlegende Untersuchungen zu einer Verbesserung der leberspezifischen Funktionen von HLCs durch Kokultur mit human umbilical vein endothelial cells (HUVECs). Ziel war es, eine Mediumzusammensetzung zu ermitteln, die sowohl die hepatische Differenzierung der hiPSCs, als auch den Erhalt der kokultivierten HUVECs unterstützt. Die Ergebnisse zeigten, dass sowohl eine Mischung aus hepatocyte culture medium (HCM) und endothelial growth medium (EGM) in einem 1:1-Verhältnis, als auch eine Mischung aus HCM und EGM-Zusätzen (ohne das EGM Basalmedium) für die HUVEC-Kultur geeignet sind. Damit wurden erfolgreich geeignete Mediumzusammensetzungen für nachfolgende Experimente zur Kokultur von hiPSCs mit HUVECs identifiziert

Haemorrhagic ulcerative duodenitis in a patient with COVID-19 infection: clinical improvement following treatment with budesonide

We present a case of a male patient in his mid-30s with COVID-19-induced lung failure requiring extracorporeal membrane oxygenation, who needed an emergency oesophagogastroduodenoscopy due to major upper gastrointestinal bleeding. Endoscopy exposed severe ulcerative duodenitis with diffuse mucosal bleeding. While CT angiography did not show any signs of ischaemia, histopathology revealed duodenitis with substantial inflammatory cell infiltrates consisting of neutrophils and CD3(+) T lymphocytes with equal CD4(+)/CD8(+) distribution. Since the composition of cell infiltrates coincides with changes in inflammatory patterns of the respiratory mucosa from patients with COVID-19 and in COVID-19-associated enterocolitis, and systemic dexamethasone treatment became standard of care in ventilated intensive care unit patients with COVID-19 infection, we initiated an individualised therapeutic attempt to treat the duodenitis with topical enteral budesonide. Follow-up oesophagogastroduodenoscopies within 4 weeks of enteral budesonide administration revealed a full clinical and histological healing of the duodenal mucosa with marked reduction of neutrophilic and lymphocytic infiltrates. To our knowledge, the current report is the first description of enteral budesonide treatment of duodenitis in a patient with COVID-19 infection and warrants further investigation, whether budesonide might constitute a novel therapeutic strategy for the management of COVID-19-related intestinal mucosal damage

Metabolism of remimazolam in primary human hepatocytes during continuous long-term infusion in a 3-D bioreactor system

Background: Remimazolam is an ultra-short acting benzodiazepine under development for procedural sedation and general anesthesia. It is hydrolyzed by CES1 to an inactive metabolite (CNS7054). Purpose: In this study, the effect of continuous remimazolam exposure on its metabolism and on CES1 expression was investigated in a dynamic 3-D bioreactor culture model inoculated with primary human hepatocytes. Methods: Remimazolam was continuously infused into bioreactors for 5 days at a final concentration of 3,000 ng/ml (6.8 μM). In parallel, 2-D cultures were run with cells from the same donors, but with discontinuous exposure to remimazolam. Results: Daily measurement of clinical chemistry parameters (glucose, lactate, urea, ammonia, and liver enzymes) in culture supernatants indicated no noxious effect of remimazolam on hepatocyte integrity as compared to untreated controls. Concentrations of remimazolam reached steady-state values of around 250 ng/ml within 8 hours in 3-D bioreactors whereas in 2-D cultures remimazolam concentrations declined to almost zero within the same time frame. Levels of CNS7054 showed an inverse time-course reaching average values of 1,350 ng/ml in perfused 3-D bioreactors resp. 2,800 ng/ml in static 2-D cultures. Analysis of mRNA expression levels of CES1 indicated no changes in gene expression over the culture period. Conclusion: The results indicated a stable metabolism of remimazolam during 5 days of continuous exposure to clinically relevant concentrations of the drug. Moreover, there was no evidence for a harmful effect of remimazolam exposure on the integrity and metabolic activity of in vitro cultivated primary human hepatocytes

Evidence for a thromboembolic pathogenesis of lung cavitations in severely ill COVID-19 patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) induces lung injury of varying severity, potentially causing severe acute respiratory distress syndrome (ARDS). Pulmonary injury patterns in COVID-19 patients differ from those in patients with other causes of ARDS. We aimed to explore the frequency and pathogenesis of cavitary lung lesions in critically ill patients with COVID-19. Retrospective study in 39 critically ill adult patients hospitalized with severe acute respiratory syndrome coronavirus 2 including lung injury of varying severity in a tertiary care referral center during March and May 2020, Berlin/Germany. We observed lung cavitations in an unusually large proportion of 22/39 (56%) COVID-19 patients treated on intensive care units (ICU), including 3/5 patients without mechanical ventilation. Median interquartile range (IQR) time between onset of symptoms and ICU admission was 11.5 (6.25-17.75) days. In 15 patients, lung cavitations were already present on the first CT scan, performed after ICU admission; in seven patients they developed during a subsequent median (IQR) observation period of 48 (35-58) days. In seven patients we found at least one cavitation with a diameter>2 cm (maximum 10 cm). Patients who developed cavitations were older and had a higher body mass index. Autopsy findings in three patients revealed that the cavitations reflected lung infarcts undergoing liquefaction, secondary to thrombotic pulmonary artery branch occlusions. Lung cavitations appear to be a frequent complication of severely ill COVID-19 patients, probably related to the prothrombotic state associated with COVID-19

Causes of death and comorbidities in hospitalized patients with COVID-19

Infection by the new corona virus strain SARS-CoV-2 and its related syndrome COVID-19 has been associated with more than two million deaths worldwide. Patients of higher age and with preexisting chronic health conditions are at an increased risk of fatal disease outcome. However, detailed information on causes of death and the contribution of pre-existing health conditions to death yet is missing, which can be reliably established by autopsy only. We performed full body autopsies on 26 patients that had died after SARS-CoV-2 infection and COVID-19 at the Charite University Hospital Berlin, Germany, or at associated teaching hospitals. We systematically evaluated causes of death and pre-existing health conditions. Additionally, clinical records and death certificates were evaluated. We report findings on causes of death and comorbidities of 26 decedents that had clinically presented with severe COVID-19. We found that septic shock and multi organ failure was the most common immediate cause of death, often due to suppurative pulmonary infection. Respiratory failure due to diffuse alveolar damage presented as immediate cause of death in fewer cases. Several comorbidities, such as hypertension, ischemic heart disease, and obesity were present in the vast majority of patients. Our findings reveal that causes of death were directly related to COVID-19 in the majority of decedents, while they appear not to be an immediate result of preexisting health conditions and comorbidities. We therefore suggest that the majority of patients had died of COVID-19 with only contributory implications of preexisting health conditions to the mechanism of death

Effects of Co-Culture Media on Hepatic Differentiation of hiPSC with or without HUVEC Co-Culture

The derivation of hepatocytes from human induced pluripotent stem cells (hiPSC) is of great interest for applications in pharmacological research. However, full maturation of hiPSC-derived hepatocytes has not yet been achieved in vitro. To improve hepatic differentiation, co-cultivation of hiPSC with human umbilical vein endothelial cells (HUVEC) during hepatic differentiation was investigated in this study. In the first step, different culture media variations based on hepatocyte culture medium (HCM) were tested in HUVEC mono-cultures to establish a suitable culture medium for co-culture experiments. Based on the results, two media variants were selected to differentiate hiPSC-derived definitive endodermal (DE) cells into mature hepatocytes with or without HUVEC addition. DE cells differentiated in mono-cultures in the presence of those media variants showed a significant increase (p &lt; 0.05) in secretion of α-fetoprotein and in activities of cytochrome P450 (CYP) isoenzymes CYP2B6 and CYP3A4 as compared with cells differentiated in unmodified HCM used as control. Co-cultivation with HUVEC did not further improve the differentiation outcome. Thus, it can be concluded that the effect of the used medium outweighed the effect of HUVEC co-culture, emphasizing the importance of the culture medium composition for hiPSC differentiation

Effect of inoculum density on human-induced pluripotent stem cell expansion in 3D bioreactors

Objective For optimized expansion of human-induced pluripotent stem cells (hiPSCs) with regards to clinical applications, we investigated the influence of the inoculum density on the expansion procedure in 3D hollow-fibre bioreactors. Materials and Methods Analytical-scale bioreactors with a cell compartment volume of 3 mL or a large-scale bioreactor with a cell compartment volume of 17 mL were used and inoculated with either 10 x 10(6) or 50 x 10(6) hiPSCs. Cells were cultured in bioreactors over 15 days; daily measurements of biochemical parameters were performed. At the end of the experiment, the CellTiter-Blue (R) Assay was used for culture activity evaluation and cell quantification. Also, cell compartment sections were removed for gene expression and immunohistochemistry analysis. Results The results revealed significantly higher values for cell metabolism, cell activity and cell yields when using the higher inoculation number, but also a more distinct differentiation. As large inoculation numbers require cost and time-extensive pre-expansion, low inoculation numbers may be used preferably for long-term expansion of hiPSCs. Expansion of hiPSCs in the large-scale bioreactor led to a successful production of 5.4 x 10(9) hiPSCs, thereby achieving sufficient cell amounts for clinical applications. Conclusions In conclusion, the results show a significant effect of the inoculum density on cell expansion, differentiation and production of hiPSCs, emphasizing the importance of the inoculum density for downstream applications of hiPSCs. Furthermore, the bioreactor technology was successfully applied for controlled and scalable production of hiPSCs for clinical use.Funding Agencies|Bundesministerium fur Bildung und Forschung [13GW0129A]</p

Microscale 3D Liver Bioreactor for In Vitro Hepatotoxicity Testing under Perfusion Conditions

The accurate prediction of hepatotoxicity demands validated human in vitro models that can close the gap between preclinical animal studies and clinical trials. In this study we investigated the response of primary human liver cells to toxic drug exposure in a perfused microscale 3D liver bioreactor. The cellularized bioreactors were treated with 5, 10, or 30 mM acetaminophen (APAP) used as a reference substance. Lactate production significantly decreased upon treatment with 30 mM APAP (p &lt; 0.05) and ammonia release significantly increased in bioreactors treated with 10 or 30 mM APAP (p &lt; 0.0001), indicating APAP-induced dose-dependent toxicity. The release of prostaglandin E2 showed a significant increase at 30 mM APAP (p &lt; 0.05), suggesting an inflammatory reaction towards enhanced cellular stress. The expression of genes involved in drug metabolism, antioxidant reactions, urea synthesis, and apoptosis was differentially influenced by APAP exposure. Histological examinations revealed that primary human liver cells in untreated control bioreactors were reorganized in tissue-like cell aggregates. These aggregates were partly disintegrated upon APAP treatment, lacking expression of hepatocyte-specific proteins and transporters. In conclusion, our results validate the suitability of the microscale 3D liver bioreactor to detect hepatotoxic effects of drugs in vitro under perfusion conditions