Streamlined Methods for Processing and Cryopreservation of Cell Therapy Products Using Automated Systems.

Streamlined procedures for processing and cryopreservation of cell therapies using good laboratory practices are integral to biomanufacturing process development and clinical applications. The protocol herein begins with the preparation of human cell types cultured as adherent (i.e., mesenchymal stromal cells, MSCs) or suspension cells (i.e., peripheral blood mononuclear cells, PBMCs) to comprehensively demonstrate procedures that are applicable to commonly used primary cell cultures. Cell processing steps consist of preparing high yields of cells for cryopreservation using instruments routinely used in cell manufacturing, including the Finia® Fill and Finish System and a controlled-rate freezer. The final steps comprise the storage of cells at subzero temperatures in liquid nitrogen vapor phase followed by the analysis of cell phenotypes before and after processing and cryopreservation, along with cell quality metrics for validation. Additionally, the protocol includes important considerations for the implementation of quality control measures for equipment operation and cell handling, as well as Good Laboratory Practices for cell manufacturing, which are essential for the translational use of cell therapies. Key features • The protocol applies to small- or large-scale manufacturing of cell therapy products. • It includes streamlined procedures for processing and cryopreservation of cells cultured as adherent cells (MSCs) and suspension cells (PBMCs). • Provides temperature control and rapid partitioning of sample in cryopreservation solution to maintain high viability of a range of cell types throughout the procedures. • This protocol employs the Finia® Fill and Finish System and a controlled-rate freezer. Graphical overview.

Differentiation and Engineering of Human Stem Cells for Smooth Muscle Generation.

Cardiovascular diseases are responsible for 31% of global deaths and are considered the main cause of death and disability worldwide. Stem cells from various sources have become attractive options for a range of cell-based therapies for smooth muscle tissue regeneration. However, for efficient myogenic differentiation, the stem cell characteristics, cell culture conditions, and their respective microenvironments need to be carefully assessed. This review covers the various approaches involved in the regeneration of vascular smooth muscles by conditioning human stem cells. This article delves into the different sources of stem cells used in the generation of myogenic tissues, the role of soluble growth factors, use of scaffolding techniques, biomolecular cues, relevance of mechanical stimulation, and key transcription factors involved, aimed at inducing myogenic differentiation. Impact statement The review article's main goal is to discuss the recent advances in the field of smooth muscle tissue regeneration. We look at various cell sources, growth factors, scaffolds, mechanical stimuli, and factors involved in smooth muscle formation. These stem cell-based approaches for vascular muscle formation will provide various options for cell-based therapies with long-term beneficial effects on patients.

Stem cell-based strategies for skeletal muscle tissue engineering.

Skeletal muscle tissue engineering has been a key area of focus over the years and has been of interest for developing regenerative strategies for injured or degenerative skeletal muscle tissue. Stem cells have gained increased attention as sources for developing skeletal muscle tissue for subsequent studies or potential treatments. Focus has been placed on understanding the molecular pathways that govern skeletal muscle formation in development to advance differentiation of stem cells towards skeletal muscle fates in vitro. Use of growth factors and transcription factors have long been the method for guiding skeletal muscle differentiation in vitro. However, further research in small molecule induced differentiation offers a xeno-free option that could result from use of animal derived factors.

Incorporating immersive learning into biomedical engineering laboratories using virtual reality.

BACKGROUND: The Covid-19 pandemic caused a sudden shift towards remote learning, moving classes to online formats. Not exempt from this switch, laboratory courses traditionally taught in-person were also moved to remote methods, costing students the opportunity to learn these skills hands-on. In order for instructors to provide course materials effectively and engagingly, non-traditional methods should be explored. Virtual reality (VR) has become more accessible in recent years. VR simulations have been used for many years as educational tools in high-risk settings such as flight or medical simulations. Immersive VR videos implemented in a remote laboratory course could provide the students with an engaging and suitable learning experience. To test the effectiveness of VR videos as a tool for remote education, VR videos of the laboratory component of a Biomolecular Engineering course were provided to students. A survey was distributed for students to self-report their experience with the videos. The survey contained quantitative and qualitative ratings of VR as an educational tool. RESULTS: The survey showed that students (~ 89% strongly agree or agree) believed the videos provided the opportunity to work at their own pace and were an appropriate length. While ~ 74% of students said that the videos provided enough information to understand the tasks, a small percentage felt that the videos improved their retention (~ 16%) and understanding (~ 9%) of the course material. About 28% of the students responded positively when asked about how VR videos improved their engagement with the material. ~ 30% reported confidence in applying the skills learned in the videos in the future and ~ 43% believe the VR videos were an acceptable alternative to in-person labs. Two-thirds of students reported feeling some form of discomfort while viewing the VR videos and 54% reported not using the headset for the videos and using the 3D video feature instead. CONCLUSIONS: As many students reported the videos containing appropriate information, the content of the videos was not an issue. A combination of improved camera quality with motion stability, more comfortable headsets, and a reduction in editing issues could greatly improve the quality and effectiveness of VR videos.

Immunomodulatory functions of human mesenchymal stromal cells are enhanced when cultured on HEP/COL multilayers supplemented with interferon-gamma.

Human mesenchymal stromal cells (hMSCs) are multipotent cells that have been proposed for cell therapies due to their immunosuppressive capacity that can be enhanced in the presence of interferon-gamma (IFN-γ). In this study, multilayers of heparin (HEP) and collagen (COL) (HEP/COL) were used as a bioactive surface to enhance the immunomodulatory activity of hMSCs using soluble IFN-γ. Multilayers were formed, via layer-by-layer assembly, varying the final layer between COL and HEP and supplemented with IFN-γ in the culture medium. We evaluated the viability, adhesion, real-time growth, differentiation, and immunomodulatory activity of hMSCs on (HEP/COL) multilayers. HMSCs viability, adhesion, and growth were superior when cultured on (HEP/COL) multilayers compared to tissue culture plastic. We also confirmed that hMSCs osteogenic and adipogenic differentiation remained unaffected when cultured in (HEP/COL) multilayers in the presence of IFN-γ. We measured the immunomodulatory activity of hMSCs by measuring the level of indoleamine 2,3-dioxygenase (IDO) expression. IDO expression was higher on (HEP/COL) multilayers treated with IFN-γ. Lastly, we evaluated the suppression of peripheral blood mononuclear cell (PBMC) proliferation when co-cultured with hMSCs on (HEP/COL) multilayers with IFN-γ. hMSCs cultured in (HEP/COL) multilayers in the presence of soluble IFN-γ have a greater capacity to suppress PBMC proliferation. Altogether, (HEP/COL) multilayers with IFN-γ in culture medium provides a potent means of enhancing and sustaining immunomodulatory activity to control hMSCs immunomodulation.

Generation and Characterization of Human Mesenchymal Stem Cell-Derived Smooth Muscle Cells.

Cardiovascular diseases are the leading cause of death worldwide. A completely autologous treatment can be achieved by using elastogenic mesenchymal stem cell (MSC)-derived smooth muscle cells (SMC) at the affected tissue site of vascular diseases such as abdominal aortic aneurysms (AAA). Thus, our work focused on evaluating the efficacy of (a) the combination of various growth factors, (b) different time periods and (c) different MSC lines to determine the treatment combination that generated SMCs that exhibited the greatest elastogenicity among the tested groups using Western blotting and flow cytometry. Additionally, total RNA sequencing was used to confirm that post-differentiation cells were upregulating SMC-specific gene markers. Results indicated that MSCs cultured for four days in PDGF + TGFß1 (PT)-infused differentiation medium showed significant increases in SMC markers and decreases in MSC markers compared to MSCs cultured without differentiation factors. RNA Seq analysis confirmed the presence of vascular smooth muscle formation in MSCs differentiated in PT medium over a seven-day period. Overall, our results indicated that origin, growth factor treatment and culture period played a major role in influencing MSC differentiation to SMCs.

Evaluation of Poly (Carbonate-Urethane) Urea (PCUU) Scaffolds for Urinary Bladder Tissue Engineering.

Although the previous success of bladder tissue engineering demonstrated the feasibility of this technology, most polyester based scaffolds used in previous studies possess inadequate mechanical properties for organs that exhibit large deformation. The present study explored the use of various biodegradable elastomers as scaffolds for bladder tissue engineering and poly (carbonate-urethane) urea (PCUU) scaffolds mimicked urinary bladder mechanics more closely than polyglycerol sebacate-polycaprolactone (PGS-PCL) and poly (ether-urethane) urea (PEUU). The PCUU scaffolds also showed cyto-compatibility as well as increased porosity with increasing stretch indicating its ability to aid in infiltration of smooth muscle cells. Moreover, a bladder outlet obstruction (BOO) rat model was used to test the safety and efficacy of the PCUU scaffolds in treating a voiding dysfunction. Bladder augmentation with PCUU scaffolds led to enhanced survival of the rats and an increase in the bladder capacity and voiding volume over a 3 week period, indicating that the high-pressure bladder symptom common to BOO was alleviated. The histological analysis of the explanted scaffold demonstrated smooth muscle cell and connective tissue infiltration. The knowledge gained in the present study should contribute towards future improvement of bladder tissue engineering technology to ultimately aide in the treatment of bladder disorders.

Nanometer-sized extracellular matrix coating on polymer-based scaffold for tissue engineering applications.

Surface modification can play a crucial role in enhancing cell adhesion to synthetic polymer-based scaffolds in tissue engineering applications. Here, we report a novel approach for layer-by-layer (LbL) fabrication of nanometer-size fibronectin and gelatin (FN-G) layers on electrospun fibrous poly(carbonate urethane)urea (PCUU) scaffolds. Alternate immersions into the solutions of fibronectin and gelatin provided thickness-controlled FN-G nano-layers (PCUU(FN-G) ) which maintained the scaffold's 3D structure and width of fibrous bundle of PCUU as evidenced by scanning electron miscroscopy. The PCUU(FN-G) scaffold improved cell adhesion and proliferation of bladder smooth muscles (BSMCs) when compared to uncoated PCUU. The high affinity of PCUU(FN-G) for cells was further demonstrated by migration of adherent BSMCs from culture plates to the scaffold. Moreover, the culture of UROtsa cells, human urothelium-derived cell line, on PCUU(FN-G) resulted in an 11-15 µm thick multilayered cell structure with cell-to-cell contacts although many UROtsa cells died without forming cell connections on PCUU. Together these results indicate that this approach will aid in advancing the technology for engineering bladder tissues in vitro. Because FN-G nano-layers formation is based on nonspecific physical adsorption of fibronectin onto polymer and its subsequent interactions with gelatin, this technique may be applicable to other polymer-based scaffold systems for various tissue engineering/regenerative medicine applications.

Tetronic(®)-based composite hydrogel scaffolds seeded with rat bladder smooth muscle cells for urinary bladder tissue engineering applications.

Natural hydrogels such as collagen offer desirable properties for tissue engineering, including cell adhesion sites, but their low mechanical strength is not suitable for bladder tissue regeneration. In contrast, synthetic hydrogels such as poly (ethylene glycol) allow tuning of mechanical properties, but do not elicit protein adsorption or cell adhesion. For this reason, we explored the use of composite hydrogel blends composed of Tetronic (BASF) 1107-acrylate (T1107A) in combination with extracellular matrix moieties collagen and hyaluronic acid seeded with bladder smooth muscle cells (BSMC). This composite hydrogel supported BSMC growth and distribution throughout the construct. When compared to the control (acellular) hydrogels, mechanical properties (peak stress, peak strain, and elastic modulus) of the cellular hydrogels were significantly greater. When compared to the 7-day time point after BSMC seeding, results of mechanical testing at the 14-day time point indicated a significant increase in both ultimate tensile stress (4.1-11.6 kPa) and elastic modulus (11.8-42.7 kPa) in cellular hydrogels. The time-dependent improvement in stiffness and strength of the cellular constructs can be attributed to the continuous collagen deposition and reconstruction by BSMC seeded in the matrix. The composite hydrogel provided a biocompatible scaffold for BSMC to thrive and strengthen the matrix; further, this trend could lead to strengthening the construct to match the mechanical properties of the bladder.