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Human mesenchymal stromal cells (hMSCs) have demonstrated, in various preclinical settings, consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation from the preclinical model into clinical practice has proven to be considerably more difficult. One key challenge being the inability to perform in situ assessment of the hMSCs in continuous culture, where the accumulation of the senescent cells impairs the culture’s viability, differentiation potential and ultimately leads to reduced therapeutic efficacies. Histochemical β -galactosidase staining is the current standard for measuring hMSC senescence, but this method is destructive and not label-free. In this study, we have investigated alternatives in quantification of hMSCs senescence, which included flow cytometry methods that are based on a combination of cell size measurements and fluorescence detection of SA- β -galactosidase activity using the fluorogenic substrate, C 12 FDG; and autofluorescence methods that measure fluorescence output from endogenous fluorophores including lipopigments. For identification of senescent cells in the hMSC batches produced, the non-destructive and label-free methods could be a better way forward as they involve minimum manipulations of the cells of interest, increasing the final output of the therapeutic-grade hMSC cultures. In this work, we have grown hMSC cultures over a period of 7 months and compared early and senescent hMSC passages using the advanced flow cytometry and autofluorescence methods, which were benchmarked with the current standard in β -galactosidase staining. Both the advanced methods demonstrated statistically significant values, (r = 0.76, p ≤ 0.001 for the fluorogenic C 12 FDG method, and r = 0.72, p ≤ 0.05 for the forward scatter method), and good fold difference ranges (1.120–4.436 for total autofluorescence mean and 1.082–6.362 for lipopigment autofluorescence mean) between early and senescent passage hMSCs. Our autofluroescence imaging and spectra decomposition platform offers additional benefit in label-free characterisation of senescent hMSC cells and could be further developed for adoption for future in situ cellular senescence evaluation by the cell manufacturers.

Patient-specific therapies require that cells be manufactured in multiple batches of small volumes, making it a challenge for conventional modes of quality control. The added complexity of inherent variability (even within batches) necessitates constant monitoring to ensure comparable end products. Hence, it is critical that new non-destructive modalities of cell monitoring be developed. Here, we study, for the first time, the use of optical spectroscopy in the determination of cellular redox across cell confluencies by exploiting the autofluorescence properties of molecules found natively within cells. This was achieved through a simple retrofitting of a standard inverted fluorescence microscope with a spectrometer output and an appropriate fluorescence filter cube. Through spectral decomposition on the acquired autofluorescence spectra, we are able to further discern the relative contributions of the different molecules, namely flavin adenine dinucleotide (FAD) and reduced nicotinamide adenine dinucleotide (NADH). This is then quantifiable as redox ratios (RR) that represent the extent of oxidation to reduction based upon the optically measured quantities of FAD and NADH. Results show that RR decreases with increasing cell confluency, which we attribute to several inter-related cellular processes. We validated the relationship between RR, metabolism and cell confluency through bio-chemical and viability assays. Live-dead and DNA damage studies were further conducted to substantiate that our measurement process had negligible effects on the cells. In this study, we demonstrate that autofluorescence spectroscopy-derived RR can serve as a rapid, non-destructive and label-free surrogate to cell metabolism measurements. This was further used to establish a relationship between cell metabolism and cellular redox across cell confluencies, and could potentially be employed as an indicator of quality in cell therapy manufacturing.

The extracellular matrix (ECM) comprises a complex milieu of proteins and other growth factors that provide mechanical, biophysical, and biochemical cues to cells. The ECM is organ specific, and its detailed composition varies across organs. Bioinks are material formulations and biological molecules or cells processed during a bioprinting process. Organ-derived decellularized ECM (dECM) bioinks have emerged as arguably the most biomimetic bioinks. Here, we review bioinks derived from different decellularized organs, the techniques used to obtain these bioinks, and the characterization methods used to evaluate their quality. We emphasize that obtaining a good-quality bioink depends on the choice of organ, animal, and decellularization method. Finally, we explore potential large-scale applications of bioinks and challenges in manufacturing such bioinks. Many individual ECM components, including collagen, fibrin, gelatin, alginate, and others, have been used as bioinks, but the natural ECM offers many physical, chemical, and biological cues that are difficult to recapitulate using only a single or just a few components. dECM bioinks could be revolutionary in terms of offering a complete biomimetic ink. dECM bioinks could be used to print more functional and relevant tissues, which would have applications for drug screening, disease modeling, and regenerative medicine. A dECM bioink is a softer material with lower mechanical strength; therefore, to ensure the integrity of the bioprinted structure, it is important to fine-tune the mechanical properties of this bioink by mixing it with either natural or synthetic materials.

The ex-vivo expansion of antigen-specific T-cells for adoptive T-cell immunotherapy requires active interaction between T-cells and antigen-presenting cells therefore culture density and environment become important variables to control. Maintenance of culture density in a static environment is traditionally performed by the expansion of the culture area through splitting of culture from a single vessel into multiple vessels—a highly laborious process. This study aims to validate the use and efficacy of a novel bioreactor, bioreactor with an expandable culture area—dual chamber (BECA-D), that was designed and developed with a cell chamber with expandable culture area (12–108 cm 2 ) and a separate media chamber to allow for in-situ scaling of culture with maintenance of optimum culture density and improved nutrient and gas exchange while minimizing disturbance to the culture. The performance of BECA-D in the culture of Epstein–Barr virus-specific T-cells (EBVSTs) was compared to the 24-well plate. BECA-D had 0.9–9.7 times the average culture yield of the 24-well plates across 5 donor sets. BECA-D was able to maintain the culture environment with relatively stable glucose and lactate levels as the culture expanded. This study concludes that BECA-D can support the culture of ex-vivo EBVSTs with lower manufacturing labour and time requirements compared to the use of the 24-well plate. BECA-D and its adaptation into a closed system with an automated platform (currently being developed) provides cell therapy manufacturers and developers with a closed scale-out solution to producing adoptive cell therapy for clinical use.

The dawn of commercial bioprinting is rapidly advancing the tissue engineering field. In the past few years, new bioprinting approaches as well as novel bioinks formulations have emerged, enabling biological research groups to demonstrate the use of such technology to fabricate functional and relevant tissue models. In recent years, several companies have launched bioprinters pushing for early adoption and democratisation of bioprinting. This article reviews the progress in commercial bioprinting since the inception, with a particular focus on the comparison of different available printing technologies and important features of the individual technologies as well as various existing applications. Various challenges and potential design considerations for next generations of bioprinters are also discussed.

[...]in-vitro 3D human tissue models would bring about the necessary complexity that may improve the reliability and accuracy of test outcomes. The approach was implemented by extruding cell-laden hyaluronic acid glycidyl methacrylate hydrogel directly into an aqueous solution containing free radicals generated by continuous blue light photo-excitation of the flavin mononucleotide/triethanolamine photo-initiator to induce diffusion-limited photo-fabrication. [...]the study has reported a novel accessible 3D tissue model platform for disease modelling and drug testing. The study evaluated the performance of 3D-printed bioresorbable airway external splint on nine different young patients with severe tracheomalacia and the results showed that the 3D printed splint not only limited the external compression and prevented airway collapse but also ensured the growth potential of the airway, making it a safe, reliable, and effective treatment for congenital heart disease patients with tracheomalacia.

Bioprinting is a promising automated platform that enables the simultaneous deposition of multiple types of cells and biomaterials to fabricate complex three-dimensional (3D) tissue constructs. Collagen-based biomaterial used in most of the previous works on skin bioprinting has poor printability and long crosslinking time. This posed an immense challenge to create 3D constructs with pre-determined shape and configuration at high throughput. Recently, the use of chitosan for wound healing applications has attracted huge attention due to its attractive traits such as its antimicrobial properties and ability to trigger hemostasis. In this paper, we optimized polyelectrolyte gelatin-chitosan hydrogel for 3D bioprinting. Modification to the chitosan was carried out via the oppositely charged functional groups from chitosan and gelatin at a specific pH of ~pH 6.5 to form polyelectrolyte complexes. The polyelectrolyte hydrogels were evaluated in terms of physical interactions within polymer blend, rheological properties (viscosities, storage and loss modulus), printing resolution at varying pressures and feed rates and biocompatibility. The polyelectrolyte gelatin-chitosan hydrogels formulated in this work was optimized for 3D bioprinting at room temperature to achieve high shape fidelity of the printed 3D constructs and good biocompatibility with fibroblast skin cells.

Technological advances have increasingly provided more and better treatment options for patients with severe burns. Here, we provide a bird’s-eye view of the product development process for third-degree burn wounds with considerations of the critical interaction with regulatory bodies, existing technological gaps, and future directions for skin substitutes. Technological advances have increasingly provided more and better treatment options for patients with severe burns. Here, we provide a bird’s-eye view of the product development process for third-degree burn wounds with considerations of the critical interaction with regulatory bodies, existing technological gaps, and future directions for skin substitutes.

Drop-on-demand (DOD) bioprinting has attracted huge attention for numerous biological applications due to its precise control over material volume and deposition pattern in a contactless printing approach. 3D bioprinting is still an emerging field and more work is required to improve the viability and homogeneity of printed cells during the printing process. Here, a general purpose bio-ink was developed using polyvinylpyrrolidone (PVP) macromolecules. Different PVP-based bio-inks (0%–3% w/v) were prepared and evaluated for their printability; the short-term and long-term viability of the printed cells were first investigated. The Z value of a bio-ink determines its printability; it is the inverse of the Ohnesorge number (Oh), which is the ratio between the Reynolds number and a square root of the Weber number, and is independent of the bio-ink velocity. The viability of printed cells is dependent on the Z values of the bio-inks; the results indicated that the cells can be printed without any significant impairment using a bio-ink with a threshold Z value of ≤9.30 (2% and 2.5% w/v). Next, the cell output was evaluated over a period of 30 min. The results indicated that PVP molecules mitigate the cell adhesion and sedimentation during the printing process; the 2.5% w/v PVP bio-ink demonstrated the most consistent cell output over a period of 30 min. Hence, PVP macromolecules can play a critical role in improving the cell viability and homogeneity during the bioprinting process.

Inertial microfluidics has emerged recently as a promising tool for high-throughput manipulation of particles and cells for a wide range of flow cytometric tasks including cell separation/filtration, cell counting, and mechanical phenotyping. Inertial focusing is profoundly reliant on the cross-sectional shape of channel and its impacts on not only the shear field but also the wall-effect lift force near the wall region. In this study, particle focusing dynamics inside trapezoidal straight microchannels was first studied systematically for a broad range of channel Re number (20 < Re < 800). The altered axial velocity profile and consequently new shear force arrangement led to a cross-lateral movement of equilibration toward the longer side wall when the rectangular straight channel was changed to a trapezoid; however, the lateral focusing started to move backward toward the middle and the shorter side wall, depending on particle clogging ratio, channel aspect ratio, and slope of slanted wall, as the channel Reynolds number further increased (Re > 50). Remarkably, an almost complete transition of major focusing from the longer side wall to the shorter side wall was found for large-sized particles of clogging ratio K ~ 0.9 (K = a/Hmin) when Re increased noticeably to ~ 650. Finally, based on our findings, a trapezoidal straight channel along with a bifurcation was designed and applied for continuous filtration of a broad range of particle size (0.3 < K < 1) exiting through the longer wall outlet with ~ 99% efficiency (Re < 100).