Acceptability of using the Raising Awareness Tool for Endometriosis (RATE) in general practice: a mixed methods pilot study.

AIMS: The Raising Awareness Tool for Endometriosis (RATE) was developed to facilitate discussions with health providers regarding endometriosis-associated symptoms. We aim to evaluate the acceptability of the RATE by general practitioners (GP), including determining the prevalence of symptoms of women presenting to general practice and immediate management of symptoms. METHODS: A mixed-methods study was undertaken using a combination of quantitative and qualitative data in Western Australian General Practices from 2021 to 2022. A purposive sample of 12 GPs were included, who recruited women (18-50 years) on attendance for consultation over a one- to two-week period, followed by qualitative interviews exploring GPs' experiences with the tool. The quantitative and qualitative components were integrated during analysis of results. RESULTS: A total of 111 women completed the RATE (mean: 33, standard deviation: 8.6 years) prior to routine consultation. The tool was considered to be acceptable for use in general practice and aided discussions on symptoms and management. Overall, 68.5% of patients experienced pelvic pain or discomfort, with 22.4% rating that this interfered with quality of life. Of those with pelvic pain, 75% had associated chronic pain conditions, and 42.1% reported allodynia. The chronic pain questions provoked GP uncertainty. After symptoms were identified, GPs arranged individualised investigations and follow-up. CONCLUSIONS: The RATE was considered to be acceptable for use in the general practice setting. It identified symptoms and initiated discussions on possible diagnosis as well as management of endometriosis. Further GP education on identifying those women at most risk of developing chronic pain syndromes is needed.

Scaling up stem cell production: harnessing the potential of microfluidic devices.

Stem cells are specialised cells characterised by their unique ability to both self-renew and transform into a wide array of specialised cell types. The widespread interest in stem cells for regenerative medicine and cultivated meat has led to a significant demand for these cells in both research and practical applications. Despite the growing need for stem cell manufacturing, the industry faces significant obstacles, including high costs for equipment and maintenance, complicated operation, and low product quality and yield. Microfluidic technology presents a promising solution to the abovementioned challenges. As an innovative approach for manipulating liquids and cells within microchannels, microfluidics offers a plethora of advantages at an industrial scale. These benefits encompass low setup costs, ease of operation and multiplexing, minimal energy consumption, and the added advantage of being labour-free. This review presents a thorough examination of the prominent microfluidic technologies employed in stem cell research and explores their promising applications in the burgeoning stem cell industry. It thoroughly examines how microfluidics can enhance cell harvesting from tissue samples, facilitate mixing and cryopreservation, streamline microcarrier production, and efficiently conduct cell separation, purification, washing, and final cell formulation post-culture.

Challenges in diagnosing and managing endometriosis in general practice: A Western Australian qualitative study.

BACKGROUND AND OBJECTIVES: To reduce diagnostic delay for women with endometriosis-related symptoms, we need to understand general practitioners' (GPs) perspectives on the challenges they face in diagnosing and managing endometriosis. METHOD: Qualitative interviews were conducted with nine Western Australian GPs to explore their knowledge, experiences and challenges with the diagnosis and management of endometriosis. RESULTS: Three themes were identified as challenges: eliciting symptoms, with subthemes of multiple and complex symptoms, clinician experience and awareness, time constraints and screening opportunities; delivering patient-centred care, with subthemes of cultural factors and health literary, perceived gender biases and women's choices and priorities; and system and service, which included learning on the job, clearer diagnostic pathways, access to services and collaborative care models. DISCUSSION: GPs can be better supported in dealing with endometriosis through raising awareness and education; recognition of endometriosis as a complex chronic condition; and the development of pragmatic guidelines, with increased access to local centres for excellent and collaborative care.

Human Mesenchymal Stem Cell Processing for Clinical Applications Using a Closed Semi-Automated Workflow.

Human mesenchymal stem cells (hMSCs) are currently being explored as a promising cell-based therapeutic modality for various diseases, with more market approvals for clinical use expected over the next few years. To facilitate this transition, addressing the bottlenecks of scale, lot-to-lot reproducibility, cost, regulatory compliance, and quality control is critical. These challenges can be addressed by closing the process and adopting automated manufacturing platforms. In this study, we developed a closed and semi-automated process for passaging and harvesting Wharton's jelly (WJ)-derived hMSCs (WJ-hMSCs) from multi-layered flasks using counterflow centrifugation. The WJ-hMSCs were expanded using regulatory compliant serum-free xeno-free (SFM XF) medium, and they showed comparable cell proliferation (population doubling) and morphology to WJ-hMSCs expanded in classic serum-containing media. Our closed semi-automated harvesting protocol demonstrated high cell recovery (~98%) and viability (~99%). The cells washed and concentrated using counterflow centrifugation maintained WJ-hMSC surface marker expression, colony-forming units (CFU-F), trilineage differentiation potential, and cytokine secretion profiles. The semi-automated cell harvesting protocol developed in the study can be easily applied for the small- to medium-scale processing of various adherent and suspension cells by directly connecting to different cell expansion platforms to perform volume reduction, washing, and harvesting with a low output volume.

The Effect of Upper Cervical Mobilization/Manipulation on Temporomandibular Joint Pain, Maximal Mouth Opening, and Pressure Pain Thresholds: A Systematic Review and Meta-Analysis.

Objective: To evaluate the efficacy of upper cervical joint mobilization and/or manipulation on reducing pain and improving maximal mouth opening (MMO) and pressure pain thresholds (PPTs) in adults with temporomandibular joint (TMJ) dysfunction compared with sham or other intervention. Data Sources: MEDLINE, CINAHL, EMBASE, and Cochrane Library from inception to June 3, 2022, were searched. Study Selection: Eight randomized controlled trials with 437 participants evaluating manual therapy (MT) vs sham and MT vs other intervention were included. Two reviewers independently extracted data and assessed risk of bias. Data Extraction: Two independent reviewers extracted information about origin, number of study participants, eligibility criteria, type of intervention, and outcome measures. Data Synthesis: Manual therapy was statistically significant in reducing pain compared with sham (mean difference [MD]: -1.93 points, 95% confidence interval [CI]: -3.61 to -0.24, P=.03), and other intervention (MD: -1.03 points, 95% CI: -1.73 to -0.33, P=.004), improved MMO compared with sham (MD: 2.11 mm, 95% CI: 0.26 to 3.96, P=.03), and other intervention (MD: 2.25 mm, 95% CI: 1.01 to 3.48, P<.001), but not statistically significant in improving PPT of masseter compared with sham (MD: 0.45 kg/cm2, 95% CI: -0.21 to 1.11, P=.18), and other intervention (MD: 0.42 kg/cm2, 95% CI: -0.19 to 1.03, P=.18), or the PPT of temporalis compared with sham (MD: 0.37 kg/cm2, 95% CI: -0.03 to 0.77, P=.07), and other intervention (MD: 0.43 kg/cm2, 95% CI: -0.60 to 1.45, P=.42). Conclusion: There appears to be limited benefit of upper cervical spine MT on TMJ dysfunction, but definitive conclusions cannot be made because of heterogeneity and imprecision of treatment effects.

Emerging Trends in Biodegradable Microcarriers for Therapeutic Applications.

Microcarriers (MCs) are adaptable therapeutic instruments that may be adjusted to specific therapeutic uses, making them an appealing alternative for regenerative medicine and drug delivery. MCs can be employed to expand therapeutic cells. MCs can be used as scaffolds for tissue engineering, as well as providing a 3D milieu that replicates the original extracellular matrix, facilitating cell proliferation and differentiation. Drugs, peptides, and other therapeutic compounds can be carried by MCs. The surface of the MCs can be altered, to improve medication loading and release, and to target specific tissues or cells. Allogeneic cell therapies in clinical trials require enormous volumes of stem cells, to assure adequate coverage for several recruitment locations, eliminate batch to batch variability, and reduce production costs. Commercially available microcarriers necessitate additional harvesting steps to extract cells and dissociation reagents, which reduces cell yield and quality. To circumvent such production challenges, biodegradable microcarriers have been developed. In this review, we have compiled key information relating to biodegradable MC platforms, for generating clinical-grade cells, that permit cell delivery at the target site without compromising quality or cell yields. Biodegradable MCs could also be employed as injectable scaffolds for defect filling, supplying biochemical signals for tissue repair and regeneration. Bioinks, coupled with biodegradable microcarriers with controlled rheological properties, might improve bioactive profiles, while also providing mechanical stability to 3D bioprinted tissue structures. Biodegradable materials used for microcarriers have the ability to solve in vitro disease modeling, and are advantageous to the biopharmaceutical drug industries, because they widen the spectrum of controllable biodegradation and may be employed in a variety of applications.

An allied reprogramming, selection, expansion and differentiation platform for creating hiPSC on microcarriers.

OBJECTIVES: Induced pluripotent stem cells (iPSCs) generated by monolayer cultures is plagued by low efficiencies, high levels of manipulation and operator unpredictability. We have developed a platform, reprogramming, expansion, and differentiation on Microcarriers, to solve these challenges. MATERIALS AND METHODS: Five sources of human somatic cells were reprogrammed, selected, expanded and differentiated in microcarriers suspension cultures. RESULTS: Improvement of transduction efficiencies up to 2 times was observed. Accelerated reprogramming in microcarrier cultures was 7 days faster than monolayer, providing between 30 and 50-fold more clones to choose from fibroblasts, peripheral blood mononuclear cells, T cells and CD34+ stem cells. This was observed to be due to an earlier induction of genes (ß-catenin, E-cadherin and EpCAM) on day 4 versus monolayer cultures which occurred on days 14 or later. Following that, faster induction and earlier stabilization of pluripotency genes occurred during the maturation phase of reprogramming. Integrated expansion without trypsinization and efficient differentiation, without embryoid bodies formation, to the three germ-layers, cardiomyocytes and haematopoietic stem cells were further demonstrated. CONCLUSIONS: Our method can solve the inherent problems of conventional monolayer cultures. It is highly efficient, cell dissociation free, can be operated with lower labor, and allows testing of differentiation efficiency without trypsinization and generation of embryoid bodies. It is also amenable to automation for processing more samples in a small footprint, alleviating many challenges of manual monolayer selection.

The Concerns and Experiences of Patients With Lumbar Spinal Stenosis Regarding Prehabilitation and Recovery After Spine Surgery: A Qualitative Study.

Objectives: To improve our understanding of patients' perspectives regarding: (1) the decision-making and prehabilitation before lumbar spinal stenosis (LSS) surgery and (2) their postoperative experiences. Design: Qualitative research with semi-structured interviews. Setting: General community. Participants: Individuals who received (N=10) and who did not receive (N=15) prehabilitation before LSS surgery were recruited at the 6-month postoperative follow-up (8 females; average age: 67.7±6.7 years) by purposive sampling. Additionally, 1 participant invited her daughter to accompany her in an interview. Interventions: Not applicable. Main Outcome Measures: Concerns and experiences of patients with LSS regarding prehabilitation and recovery after spine surgery. Results: Thematic analysis was conducted to identify 4 themes inductively: (1) sources of information about LSS surgery; (2) factors affecting the surgical decision-making; (3) attitudes toward prehabilitation; and (4) postoperative recovery. All participants desired to have more preoperative education to inform their surgical decision-making. There were mixed opinions regarding the perceived benefits of prehabilitation because some individuals hesitated to participate in prehabilitation because of their symptoms, or the cost or time of traveling. Many participants expected some or even complete relief of LSS-related symptoms after surgery. However, not all participants experienced the expected postoperative improvements. Some participants only experienced temporary symptomatic relief, while others experienced new postoperative symptoms. Patients generally found that postoperative exercises taught by physiotherapists were useful although their compliance decreased over time. Conclusions: Our study highlights the need for better preoperative LSS education. Because face-to-face prehabilitation or postoperative rehabilitation may not be feasible for all patients, future studies should explore whether online-based prehabilitation or postoperative rehabilitation may benefit certain patient subgroups.

Wrist accelerometry for monitoring dementia agitation behaviour in clinical settings: A scoping review.

Agitated behaviour among elderly people with dementia is a challenge in clinical management. Wrist accelerometry could be a versatile tool for making objective, quantitative, and long-term assessments. The objective of this review was to summarise the clinical application of wrist accelerometry to agitation assessments and ways of analysing the data. Two authors independently searched the electronic databases CINAHL, PubMed, PsycInfo, EMBASE, and Web of Science. Nine (n = 9) articles were eligible for a review. Our review found a significant association between the activity levels (frequency and entropy) measured by accelerometers and the benchmark instrument of agitated behaviour. However, the performance of wrist accelerometry in identifying the occurrence of agitation episodes was unsatisfactory. Elderly people with dementia have also been monitored in existing studies by investigating the at-risk time for their agitation episodes (daytime and evening). Consideration may be given in future studies on wrist accelerometry to unifying the parameters of interest and the cut-off and measurement periods, and to using a sampling window to standardise the protocol for assessing agitated behaviour through wrist accelerometry.

Getting square pegs out through round holes: A survey of Australian and New Zealand Gynaecologists regarding specimen retrieval.

AIMS: To evaluate morcellation practices among Fellows of the Royal Australian and New Zealand College of Obstetricians and Gynaecologists (RANZCOG). MATERIALS AND METHODS: RANZCOG Fellows were invited to complete an online survey. This anonymous, cross-sectional survey consisted of 29 questions regarding demographics and morcellation practices. RESULTS: Four hundred and thirty eight (19.04%) of 2300 RANZCOG Fellows responded, and of these 258 (11.22%) completed the entire survey; analysis was undertaken on data from the latter respondents. Respondents were broadly representative of all RANZCOG Fellows regarding gender, age, and location. Of the respondents, 53.10% considered themselves advanced laparoscopic surgeons. Of respondents who had worked as gynaecology consultants prior to 2014, 39.39% used uncontained power morcellation prior to 2014, compared to 17.58% since (a decrease of 44.63%). The most common reasons for utilising uncontained power morcellation less often were the 2014 Food and Drug Administration warnings (40.31%), risk of adverse outcomes (33.72%), and recommendations from colleges such as RANZCOG (27.13%). When undertaking an operation that required specimen extraction, the most common methods used were: employing an open approach from the get-go (utilised by respondents in 31.01% of such cases); contained manual morcellation (28.90%); and conversion to intra-operative laparotomy (10.10%). CONCLUSIONS: There has been a strong trend away from uncontained power morcellation since 2014, with a 36.00% increase in clinicians who never use uncontained power morcellation, and an 80.65% decrease in clinicians who always use this method of specimen extraction. The most common reason cited for employing uncontained power morcellation less often was the 2014 Food and Drug Administration's warnings.

Early Tibial Component Fractures in a Cementless, 3D-Printed, Titanium Implant.

Fracture of the tibial baseplate in total knee arthroplasty is a rare occurrence, particularly in short- and mid-term follow-up. This case series documents the first known report in the literature of fatigue fracture of a cementless, 3D-printed, highly porous titanium tibial component. We recommend regular follow-up visits with radiographs to confirm adequate total knee arthroplasty component positioning and alignment.

Selection of O-negative induced pluripotent stem cell clones for high-density red blood cell production in a scalable perfusion bioreactor system.

OBJECTIVES: Large-scale generation of universal red blood cells (RBCs) from O-negative (O-ve) human induced pluripotent stem cells (hiPSCs) holds the potential to alleviate worldwide shortages of blood and provide a safe and secure year-round supply. Mature RBCs and reticulocytes, the immature counterparts of RBCs generated during erythropoiesis, could also find important applications in research, for example in malaria parasite infection studies. However, one major challenge is the lack of a high-density culture platform for large-scale generation of RBCs in vitro. MATERIALS AND METHODS: We generated 10 O-ve hiPSC clones and evaluated their potential for mesoderm formation and erythroid differentiation. We then used a perfusion bioreactor system to perform studies with high-density cultures of erythroblasts in vitro. RESULTS: Based on their tri-lineage (and specifically mesoderm) differentiation potential, we isolated six hiPSC clones capable of producing functional erythroblasts. Using the best performing clone, we demonstrated the small-scale generation of high-density cultures of erythroblasts in a perfusion bioreactor system. After process optimization, we were able to achieve a peak cell density of 34.7 million cells/ml with 92.2% viability in the stirred bioreactor. The cells expressed high levels of erythroblast markers, showed oxygen carrying capacity, and were able to undergo enucleation. CONCLUSIONS: This study demonstrated a scalable platform for the production of functional RBCs from hiPSCs. The perfusion culture platform we describe here could pave the way for large volume-controlled bioreactor culture for the industrial generation of high cell density erythroblasts and RBCs.

Strategies to enhance immunomodulatory properties and reduce heterogeneity in mesenchymal stromal cells during ex vivo expansion.

Therapies using mesenchymal stromal cells (MSCs) to treat immune and inflammatory conditions are now at an exciting stage of development, with many MSC-based products progressing to phase II and III clinical trials. However, a major bottleneck in the clinical translation of allogeneic MSC therapies is the variable immunomodulatory properties of MSC products due to differences in their tissue source, donor heterogeneity and processes involved in manufacturing and banking. This variable functionality of MSC products likely contributes to the substantial inconsistency observed in the clinical outcomes of phase III trials of MSC therapies; several trials have failed to reach the primary efficacy endpoint. In this review, we discuss various strategies to consistently maintain or enhance the immunomodulatory potency of MSCs during ex vivo expansion, which will enable the manufacture of allogeneic MSC banks that have high potency and low variability. Biophysical and biochemical priming strategies, the use of culture additives such as heparan sulfates, and genetic modification can substantially enhance the immunomodulatory properties of MSCs during in vitro expansion. Furthermore, robust donor screening, the use of biomarkers to select for potent MSC subpopulations, and rigorous quality testing to improve the release criteria for MSC banks have the potential to reduce batch-to-batch heterogeneity and enhance the clinical efficacy of the final MSC product. Machine learning approaches to develop predictive models of individual patient response can enable personalized therapies and potentially establish correlations between in vitro potency measurements and clinical outcomes in human trials.

Integrating Human-Induced Pluripotent Stem Cell Expansion Capability and Cardiomyocyte Differentiation Potential in a Microcarrier Suspension Culture.

Human-induced pluripotent stem cells are known for their high proliferation capacity as well as their ability to differentiate to different lineages (Ban et al., Theranostics 7(7):2067-2077, 2017; Chen et al., Stem Cell Res 15(2):365-375, 2015; Serra et al., Trends Biotechnol 30(6):350-359, 2012). For stem-cell-derived cardiomyocytes to evolve into a scalable therapeutic source, a large quantity of highly pure cardiomyocytes is needed. Thus, lies the challenge of defining an efficient cardiomyocyte differentiation process. This chapter describes a method to evaluate multiple human-induced pluripotent stem cell lines for their cardiac differentiation potentials before evaluating their integrated proliferation and differentiation abilities in microcarrier cultures in a spinner culture format.

Gamma Delta T Cells and Their Involvement in COVID-19 Virus Infections.

The global outbreak of the SARS-Cov-2 virus in 2020 has killed millions of people worldwide and forced large parts of the world into lockdowns. While multiple vaccine programs are starting to immunize the global population, there is no direct cure for COVID-19, the disease caused by the SARS-Cov-2 infection. A common symptom in patients is a decrease in T cells, called lymphopenia. It is as of yet unclear what the exact role of T cells are in the immune response to COVID-19. The research so far has mainly focused on the involvement of classical αß T cells. However, another subset of T cells called γδ T cells could have an important role to play. As part of the innate immune system, γδ T cells respond to inflammation and stressed or infected cells. The γδ T cell subset appears to be particularly affected by lymphopenia in COVID-19 patients and commonly express activation and exhaustion markers. Particularly in children, this subset of T cells seems to be most affected. This is interesting and relevant because γδ T cells are more prominent and active in early life. Their specific involvement in this group of patients could indicate a significant role for γδ T cells in this disease. Furthermore, they seem to be involved in other viral infections and were able to kill SARS infected cells in vitro. γδ T cells can take up, process and present antigens from microbes and human cells. As e.g. tumour-associated antigens are presented by MHC on γδ T cells to classical T-cells, we argue here that it stands to reason that also viral antigens, such as SARS-Cov-2-derived peptides, can be presented in the same way. γδ T cells are already used for medical purposes in oncology and have potential in cancer therapy. As γδ T cells are not necessarily able to distinguish between a transformed and a virally infected cell it could therefore be of great interest to investigate further the relationship between COVID-19 and γδ T cells.

Psychological Effects of Screen Time in Health Care Workers During the COVID-19 Pandemic.

Objective: As the coronavirus disease (COVID-19) outbreak is the first pandemic to occur in the modern smartphone era, people universally rely on their electronic devices to stay current on the rapidly evolving circumstances. The objective of this study was to examine how daily screen time levels affect the mental health of health care workers attempting to stay up to date on the ever-changing COVID-19-related information available to them.Methods: Health care workers at an academic teaching hospital were asked to participate in a 12-question online-based survey between the dates of May 30, 2020, and June 3, 2020. The questions included their sex, age range, occupation, department, daily screen time, changes in screen time in the last 4 weeks, and mental health outcomes such as sleep, mood, anxiety, and difficulty controlling worry.Results: No association was found between age, sex, occupation, and screen time. There was a statistically significant association between the type of department and daily screen time hours (P = .012). A positive trend was noted between screen time and sleep disruption (P = .09). An increase in hours in the last 4 weeks was associated with age (P = .03). A positive trend was also noted for an increase in screen hours and sleep disruption (P = .11) and anxiety (P = .10).Conclusions: A possible explanation for our finding of screen time not being associated with mental health outcomes could be that the knowledge that information was readily available through technology provided comfort to people as the pandemic evolved and brought changes to their daily lives.

Uterine leiomyoma is associated with the risk of developing endometriosis: A nationwide cohort study involving 156,195 women.

OBJECTIVE: Evidence for an association between uterine leiomyoma and increased risk of endometriosis is limited by small sample sizes and short follow-up periods. We assessed this association in a large nationwide sample with 14 years of data. DESIGN: Data were sourced from Taiwan's Longitudinal Health Insurance Database 2000 (LHID2000). MATERIALS AND METHODS: We identified 31,239 women aged ≥20 years diagnosed with uterine leiomyoma (International Classification of Disease, Ninth Revision, Clinical Modification [ICD-9-CM] code 218) between Jan 1, 2000 and Dec 31, 2012, who were matched with 124,956 controls (1:4) by 5-year age groups and year of diagnosis. Follow-up was from the date of LHID2000 entry to the first occurrence of endometriosis, loss to follow-up, insurance termination, or until December 31, 2013, whichever was earlier. RESULTS: In Cox regression analysis, the adjusted hazard ratio (aHR) for endometriosis in women with uterine leiomyoma was 6.44 (95% CI, 6.18, 6.72) compared with controls. The risk of endometriosis was significantly increased in women with uterine leiomyoma and comorbidities of tube-ovarian infection (aHR 2.86; 95% CI, 1.28, 6.36), endometritis (1.14; 1.06, 1.24), infertility (1.26; 1.16, 1.37), or allergic diseases (1.11; 1.05, 1.17). Having both uterine leiomyoma and endometritis significantly increased the risk of endometriosis (aHR 6.73; 95% CI, 6.07, 7.45) versus having only uterine leiomyoma (6.61; 6.33, 6.91) or endometritis (1.49; 1.31, 1.69). Similarly, having both uterine leiomyoma and infertility significantly increased the risk of endometriosis (aHR 6.95; 95% CI, 6.21, 7.78) versus having only uterine leiomyoma (6.66; 6.38, 6.96) or infertility (1.78; 1.57, 2.02). CONCLUSIONS: A diagnosis of uterine leiomyoma appears to increase the risk of endometriosis. Patients presenting with uterine fibroids should be encouraged to give informed consent for possible simultaneous surgical treatment of endometriosis.

Multiomics analyses of cytokines, genes, miRNA, and regulatory networks in human mesenchymal stem cells expanded in stirred microcarrier-spinner cultures.

Mesenchymal stem cells (MSCs) are of great clinical interest as a form of allogenic therapy due to their excellent regenerative and immunomodulatory effects for various therapeutic indications. Stirred suspension bioreactors using microcarriers (MC) have been used for large-scale production of MSCs compared to planar cultivation systems. Previously, we have demonstrated that expansion of MSCs in MC-spinner cultures improved chondrogenic, osteogenic, and cell migration potentials as compared to monolayer-static cultures. In this study, we sought to address this by analyzing global gene expression patterns, miRNA profiles and secretome under both monolayer-static and MC-spinner cultures in serum-free medium at different growth phases. The datasets revealed differential expression patterns that correlated with potentially improved MSC properties in cells from MC-spinner cultures compared to those of monolayer-static cultures. Transcriptome analysis identified a unique expression signature for cells from MC-spinner cultures, which correlated well with miRNA expression, and cytokine secretion involved in key MSC functions. Importantly, MC-spinner cultures and conditioned medium showed increased expression of factors that possibly enhance pathways of extracellular matrix dynamics, cellular metabolism, differentiation potential, immunoregulatory function, and wound healing. This systematic analysis provides insights for the efficient optimization of stem cell bioprocessing and infers that MC-based bioprocess manufacturing could improve post-expansion cellular properties for stem cell therapies.

A Scalable Suspension Platform for Generating High-Density Cultures of Universal Red Blood Cells from Human Induced Pluripotent Stem Cells.

Universal red blood cells (RBCs) differentiated from O-negative human induced pluripotent stem cells (hiPSCs) could find applications in transfusion medicine. Given that each transfusion unit of blood requires 2 trillion RBCs, efficient bioprocesses need to be developed for large-scale in vitro generation of RBCs. We have developed a scalable suspension agitation culture platform for differentiating hiPSC-microcarrier aggregates into functional RBCs and have demonstrated scalability of the process starting with 6 well plates and finally demonstrating in 500 mL spinner flasks. Differentiation of the best-performing hiPSCs generated 0.85 billion erythroblasts in 50 mL cultures with cell densities approaching 1.7 × 107 cells/mL. Functional (oxygen binding, hemoglobin characterization, membrane integrity, and fluctuations) and transcriptomics evaluations showed minimal differences between hiPSC-derived and adult-derived RBCs. The scalable agitation suspension culture differentiation process we describe here could find applications in future large-scale production of RBCs in controlled bioreactors.

Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor.

BACKGROUND: The production of large quantities of cardiomyocyte is essential for the needs of cellular therapies. This study describes the selection of a human-induced pluripotent cell (hiPSC) line suitable for production of cardiomyocytes in a fully integrated bioprocess of stem cell expansion and differentiation in microcarrier stirred tank reactor. METHODS: Five hiPSC lines were evaluated first for their cardiac differentiation efficiency in monolayer cultures followed by their expansion and differentiation compatibility in microcarrier (MC) cultures under continuous stirring conditions. RESULTS: Three cell lines were highly cardiogenic but only one (FR202) of them was successfully expanded on continuous stirring MC cultures. FR202 was thus selected for cardiac differentiation in a 22-day integrated bioprocess under continuous stirring in a stirred tank bioreactor. In summary, we integrated a MC-based hiPSC expansion (phase 1), CHIR99021-induced cardiomyocyte differentiation step (phase 2), purification using the lactate-based treatment (phase 3) and cell recovery step (phase 4) into one process in one bioreactor, under restricted oxygen control (< 30% DO) and continuous stirring with periodic batch-type media exchanges. High density of undifferentiated hiPSC (2 ± 0.4 × 106 cells/mL) was achieved in the expansion phase. By controlling the stirring speed and DO levels in the bioreactor cultures, 7.36 ± 1.2 × 106 cells/mL cardiomyocytes with > 80% Troponin T were generated in the CHIR99021-induced differentiation phase. By adding lactate in glucose-free purification media, the purity of cardiomyocytes was enhanced (> 90% Troponin T), with minor cell loss as indicated by the increase in sub-G1 phase and the decrease of aggregate sizes. Lastly, we found that the recovery period is important for generating purer and functional cardiomyocytes (> 96% Troponin T). Three independent runs in a 300-ml working volume confirmed the robustness of this process. CONCLUSION: A streamlined and controllable platform for large quantity manufacturing of pure functional atrial, ventricular and nodal cardiomyocytes on MCs in conventional-type stirred tank bioreactors was established, which can be further scaled up and translated to a good manufacturing practice-compliant production process, to fulfill the quantity requirements of the cellular therapeutic industry.