The development of bioprocesses capable of producing large numbers of human induced pluripotent stem cells (hiPSC) in a robust and safe manner is critical for the application of these cells in biotechnological and medical applications. Scalable expansion of hiPSC is often performed using polystyrene microcarriers, which have to be removed from the cell suspension using a separation step that causes loss of viable cells. In this study, application of novel xeno‐free dissolvable microcarriers (DM) for an efficient and integrated expansion and harvesting of hiPSC is demonstrated. After an initial screening under static conditions, hiPSC culture using DM is performed in dynamic culture, using spinner‐flasks. A maximum 4.0 ± 0.8‐fold expansion is achieved after 5 days of culture. These results are validated with a second cell line and the culture is successfully adapted to fully xeno‐free conditions. Afterwards, cell recovery is made within the spinner flask, being obtained a 92 ± 4% harvesting yield, which is significantly higher than the one obtained for the conventional filtration‐based method (45 ± 3%). Importantly, the expanded and harvested hiPSC maintain their pluripotency and multilineage differentiation potential. The results here described represent a significant improvement of the downstream processing after microcarrier‐based hiPSC expansion, leading to a more cost‐effective and efficient bioprocess.

The COVID-19 pandemic is a major human health concern. The pathogen responsible for COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), invades its host through the interaction of its spike (S) protein with a host cell receptor, angiotensin-converting enzyme 2 (ACE2). In addition to ACE2, heparan sulfate (HS) on the surface of host cells also plays a significant role as a co-receptor. Our previous studies demonstrated that sulfated glycans, such as heparin and fucoidans, show anti-COVID-19 activities. In the current study, rhamnan sulfate (RS), a polysaccharide with a rhamnose backbone from a green seaweed, Monostroma nitidum, was evaluated for binding to the S-protein from SARS-CoV-2 and inhibition of viral infectivity in vitro. The structural characteristics of RS were investigated by determining its monosaccharide composition and performing two-dimensional nuclear magnetic resonance. RS inhibition of the interaction of heparin, a highly sulfated HS, with the SARS-CoV-2 spike protein (from wild type and different mutant variants) was studied using surface plasmon resonance (SPR). In competitive binding studies, the IC50 of RS against the S-protein receptor binding domain (RBD) binding to immobilized heparin was 1.6 ng/mL, which is much lower than the IC50 for heparin (~750 ng/mL). RS showed stronger inhibition than heparin on the S-protein RBD or pseudoviral particles binding to immobilized heparin. Finally, in an in vitro cell-based assay, RS showed strong antiviral activities against wild type SARS-CoV-2 and the delta variant.

With the increased prevalence of new SARS-CoV-2 variants of concern, such as Delta and Omicron, the COVID-19 pandemic has become an ongoing human health disaster, killing millions worldwide. SARS-CoV-2 invades its host through the interaction of its spike (S) protein with a host cell receptor, angiotensin-converting enzyme 2 (ACE2). In addition, heparan sulfate (HS) on the surface of host cells plays an important role as a co-receptor for this viral pathogen–host cell interaction. Our previous studies demonstrated that many sulfated glycans, such as heparin, fucoidans, and rhamnan sulfate have anti-SARS-CoV-2 activities. In the current study, a small library of sulfated glycans and highly negatively charged compounds, including pentosan polysulfate (PPS), mucopolysaccharide polysulfate (MPS), sulfated lactobionic acid, sulodexide, and defibrotide, was assembled and evaluated for binding to the S-proteins and inhibition of viral infectivity in vitro. These compounds inhibited the interaction of the S-protein receptor-binding domain (RBD) (wild type and different variants) with immobilized heparin, a highly sulfated HS, as determined using surface plasmon resonance (SPR). PPS and MPS showed the strongest inhibition of interaction of heparin and S-protein RBD. The competitive binding studies showed that the IC50 of PPS and MPS against the S-protein RBD binding to immobilized heparin was ~35 nM and ~9 nM, respectively, much lower than the IC50 for soluble heparin (IC50 = 56 nM). Both PPS and MPS showed stronger inhibition than heparin on the S-protein RBD or spike pseudotyped lentiviral particles binding to immobilized heparin. Finally, in an in vitro cell-based assay, PPS and MPS exhibited strong antiviral activities against pseudotyped viral particles of SARS-CoV-2 containing wild-type or Delta S-proteins.

Three-dimensional (3D) cell culture methods have been widely used on a range of cell types, including stem cells to modulate precisely the cellular biophysical and biochemical microenvironment and control various cell signaling cues. As a result, more in vivo -like microenvironments are recapitulated, particularly through the formation of multicellular spheroids and organoids, which may yield more valid mechanisms of disease. Recently, genome-engineering tools such as CRISPR Cas9 have expanded the repertoire of techniques to control gene expression, which complements external signaling cues with intracellular control elements. As a result, the combination of CRISPR Cas9 and 3D cell culture methods enhance our understanding of the molecular mechanisms underpinning several disease phenotypes and may lead to developing new therapeutics that may advance more quickly and effectively into clinical candidates. In addition, using CRISPR Cas9 tools to rescue genes brings us one step closer to its use as a gene therapy tool for various degenerative diseases. Herein, we provide an overview of bridging of CRISPR Cas9 genome editing with 3D spheroid and organoid cell culture to better understand disease progression in both patient and non-patient derived cells, and we address potential remaining gaps that must be overcome to gain widespread use.

SARS-CoV-2 receptor binding domains (RBDs) interact with both the ACE2 receptor and heparan sulfate on the surface of host cells to enhance SARS-CoV-2 infection. We show that suramin, a polysulfated synthetic drug, binds to the ACE2 receptor and heparan sulfate binding sites on the RBDs of wild-type, Delta, and Omicron variants. Specifically, heparan sulfate and suramin had enhanced preferential binding for Omicron RBD, and suramin is most potent against the live SARS-CoV-2 Omicron variant (B.1.1.529) when compared to wild type and Delta (B.1.617.2) variants in vitro. These results suggest that inhibition of live virus infection occurs through dual SARS-CoV-2 targets of S-protein binding and previously reported RNA-dependent RNA polymerase inhibition and offers the possibility for this and other polysulfated molecules to be used as potential therapeutic and prophylactic options against COVID-19.

In this manuscript, we employ parallel batch stability and chromatographic screens in concert with linear and step gradient experiments to develop a high yield, HCP clearance anion exchange capture process for lentiviral vector (LVV) purification. An initial broad resin screen is carried out to determine anion exchange‐based resins that exhibit high recovery of LVV. LVV stability is then evaluated and conditions are established where the vector exhibits good stability, namely phosphate buffer at pH 6.5–7.5, with low to moderate salt concentrations. A subsequent high‐throughput batch screen is then carried out with a subset of resins selected from the first screen under stable conditions to identify optimal wash and elution steps to further improve product yield and protein clearance. Linear gradient experiments are also conducted in mini‐column format to refine the operating conditions and final step gradient processes are established that exhibit greater than 70% yield of infectious LVV while also achieving up to 2.89 log reduction values (LRV) of HCPs during the process. The large set of stability and chromatographic data provided in this work represent an important contribution to knowledge in the field about the chromatographic efficacy of a wide range of resins for LVV bioprocessing under stable conditions.

Tissue engineering and stem cell research greatly benefit from cell encapsulation within hydrogels as it promotes cell expansion and differentiation. Affinity-triggered hydrogels, an appealing solution for mild cell encapsulation, rely on selective interactions between the ligand and target and also on the multivalent presentation of these two components. Although these hydrogels represent a versatile option to generate dynamic, tunable, and highly functional materials, the design of hydrogel properties based on affinity and multivalency remains challenging and unstudied. Here, the avidin–biotin affinity pair, with the highest reported affinity constant, is used to address this challenge. It is demonstrated that the binding between the affinity hydrogel components is influenced by the multivalent display selected. In addition, the natural multivalency of the interaction must be obeyed to yield robust multicomponent synthetic protein hydrogels. The hydrogel’s resistance to erosion depends on the right stoichiometric match between the hydrogel components. The developed affinity-triggered hydrogels are biocompatible and support encapsulation of induced pluripotent stem cells and their successful differentiation into a neural cell line. This principle can be generalized to other affinity pairs using multimeric proteins, yielding biomaterials with controlled performance.

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