Stem Cell Culture Research Articles

Considerations for oral and dental tissues in holistic care during long-haul space flights.

The health of astronauts during and after the return from long-haul space missions is paramount. There is plethora of research in the literature about the medical side of astronauts' health, however, the dental and oral health of the space crew seem to be overlooked with limited information in the literature about the effects of the space environment and microgravity on the oral and dental tissues. In this article, we shed some light on the latest available research related to space dentistry and provide some hypotheses that could guide the directions of future research and help maintain the oral health of space crews. We also promote for the importance of regenerative medicine and dentistry as well highlight the opportunities available in the expanding field of bioprinting/biomanufacturing through utilizing the effects of microgravity on stem cells culture techniques. Finally, we provide recommendations for adopting a multidisciplinary approach for oral healthcare during long-haul space flights.

Compact lens-free imager using a thin-film transistor for long-term quantitative monitoring of stem cell culture and cardiomyocyte production.

With advancements in human induced pluripotent stem cell (hiPSC) technology, there is an increasing demand for quality control techniques to manage the long-term process of target cell production effectively. While monitoring systems designed for use within incubators are promising for assessing culture quality, existing systems still face challenges in terms of compactness, throughput, and available metrics. To address these limitations, we have developed a compact and high-throughput lens-free imaging device named INSPCTOR. The device is as small as a standard culture plate, which allows for the installation of multiple units within an incubator. INSPCTOR utilises a large thin-film transistor image sensor, enabling simultaneous observation of six independent culture environments, each approximately 1 cm2. With this device, we successfully monitored the confluency of hiPSC cultures and identified the onset timing of epithelial-to-mesenchymal transition during mesodermal induction. Additionally, we quantified the beating frequency and conduction of hiPSC-derived cardiomyocytes by using high-speed imaging modes. This enabled us to identify the onset of spontaneous beating during differentiation and assess chronotropic responses in drug evaluations. Moreover, by tracking beating frequency over 10 days of cardiomyocyte maturation, we identified week-scale and daily-scale fluctuations, the latter of which correlated with cellular metabolic activity. The metrics derived from this device would enhance the reproducibility and quality of target cell production.

Standardized GMP-Compliant Scalable 3D-Bioprocessing of Epidermal Stem Cells for Diabetic Foot Ulcers.

Diabetic foot ulcers (DFUs) pose a significant threat to the health and well-being of individuals with diabetes, often leading to lower limb amputations. Fortunately, epidermal stem cell therapy offers hope for improving the treatment of DFUs. By leveraging 3D culture techniques, the scalability of stem cell manufacturing can be greatly enhanced. In particular, using bioactive materials and scaffolds can promote the healing potential of cells, enhance their proliferation, and facilitate their survival. Furthermore, 3D tissue-mimicking cultures can accurately replicate the complex interactions between cells and extracellular matrix, thereby ensuring that the stem cells are primed for therapeutic application. To ensure the safety and quality of these stem cells, it is essential to adhere to good manufacturing practice (GMP) principles during cultivation. This chapter provides a comprehensive overview of the step-by-step process for GMP-based 3D epidermal stem cell cultivation, thus laying the groundwork for developing reliable regenerative medicine therapies.

Platelet-Rich Plasma as Xenofree-Growth Factor For Mesenchymal Stem Cells Culture

Stem cell-based therapy is currently believed to be the future of medicine. The type of stem cell that is widely used in cell-based therapy is mesenchymal stem cells (MSCs). In stem cell-based therapy, the MSCs that will be used mostly need to be expanded first by culturing the cells. The serum that is often used in MSCs culturing is a fetal bovine serum (FBS). However, animal components should be avoided in clinical applications and Good Manufacturing Practices (GMP). FBS is not adhering to GMP, which compromises the safety and effectiveness of cell-based therapies. Therefore, other supplements are required for cultures without animal serum constituents. Platelet-rich plasma (PRP) is a plasma fraction derived from blood with a platelet concentration of 3-5 times higher than in blood. Several studies have shown that PRP contains various growth factors that can increase the proliferative and differentiation capacity of MSCs which is equivalent to or even better than FBS. PRP also has the advantage that it can be an autologous or allogeneic product from human peripheral blood, so it is free from animal substances. Therefore in the future PRP has great potential as an alternative to FBS both in culture expansion and in MSCs processing.

Equine Induced Pluripotent Stem Cell Culture.

Groundbreaking work by Takahashi and Yamanaka in 2006 demonstrated that non-embryonic cells can be reprogrammed into pluripotent stem cells (PSCs) by forcing the expression of a defined set of transcription factors in culture, thus overcoming ethical concerns linked to embryonic stem cells. Induced PSCs have since revolutionized biomedical research, holding tremendous potential also in other areas such as livestock production and wildlife conservation. iPSCs exhibit broad accessibility, having been derived from a multitude of cell types and species. Apart from humans, iPSCs hold particular medical promise in the horse. The potential of iPSCs has been shown in a variety of biomedical contexts in the horse. However, progress in generating therapeutically useful equine iPSCs has lagged behind that reported in humans, with the generation of footprint-free iPSCs using non-integrative reprogramming approaches having proven particularly challenging. A greater understanding of the underlying molecular pathways and essential factors required for the generation and maintenance of equine iPSCs and their differentiation into relevant lineages will be critical for realizing their significant potential in veterinary regenerative medicine. This article outlines up-to-date protocols for the successful culture of equine iPSC, including colony selection, expansion, and adaptation to feeder-free conditions.

Conditioned medium of IGF-1-induced human synovial membrane mesenchymal stem cells effects on inflammatory response of osteoarthritic in vitro model

Synovial membrane mesenchymal stem cells (SM-MSCs) possess excellent regenerative potential, making them a viable option for osteoarthritis (OA) therapy. Moreover, these cells can be easily obtained from the human body. Insulin-like growth factor (IGF-1) can be used to decrease the pro-inflammatory marker, these markers can trigger inflammation in OA. These challenges can be addressed by utilizing a conditioned medium (CM) derived from stem cell culture. This study aims to compare the effective way of OA therapy between CM SM-MSC and CM SM-MSC induced by IGF-1 by observe the pro-inflammatory markers such as NF-kB, RANTES, PGE2, COL1 and the anti-inflammatory marker, Aggrecan (ACAN). Chondrocyte cells induced IL-1β as OA model (IL1β-CHON002) treated with IGF-1 15% and 30% and without IGF1-induced SM-MSCs-CM to know its effectiveness in decreasing the pro-inflammatory markers. ELISA and RT-qPCR methods were performed to analyze this effect. Based on the data result, SM-MSCs-CM is estimated to have the potential to treat OA as seen from the content of growth factors in CM, decreasing the markers of inflammation. The most significant reduction in PGE2, RANTES, NF-kB, and COL1 concentration was found in the treatment of SM-MSCs-CM cultured with IGF1 concentrations of 150 ng/mL. The higher aggrecan (ACAN) concentration was found in IGF150 15%. Conclusion: CM from SM-MSCs cultured with IGF150 with concentrations of 15% and 30% resulted in the most effective concentration to decrease the concentration of pro-inflammatory markers and also to increase the anti-inflammatory markers.

Functional and Molecular Heterogeneity in Glioma Stem Cells Derived from Multiregional Sampling.

Glioblastoma (GBM) is an aggressive and highly heterogeneous primary brain tumor. Glioma stem cells represent a subpopulation of tumor cells with stem cell traits that are presumed to be the cause of tumor relapse. There exists complex tumor heterogeneity in drug sensitivity patterns between glioma stem cell (GSC) cultures derived from different patients. Here, we describe that heterogeneity also exists between GSC cultures derived from multiple biopsies within a single tumor. From biopsies harvested within spatially distinct regions representing the entire tumor mass, we established seven GSC cultures and compared their stem cell properties, mutations, gene expression profiles, and drug sensitivity patterns against 115 different anticancer drugs. The results were compared to 14 GSC cultures derived from other patients. Between the multiregional-derived GSC cultures, we observed only minor differences in their phenotype, proliferative capacity, and global gene expression. Further, they displayed intratumoral heterogeneity in mutational profiles and sensitivity patterns to anticancer drugs. This heterogeneity, however, did not exceed the extensive heterogeneity found between GSC cultures derived from other GBM patients. Our results suggest that the use of GSC cultures from one single focal biopsy may underestimate the overall complexity of the GSC population and display the importance of including GSC cultures reflecting the entire tumor mass in drug screening strategies.

Identification of RNA-based cell-type markers for stem-cell manufacturing systems with a statistical scoring function

Cell-type biomarkers are useful in stem-cell manufacturing to monitor cell purity, quantity, and quality. However, the study on cell-type markers, specifically for stem cell manufacture, is limited. Emerging questions include which RNA transcripts can serve as biomarkers during stem cell culture and how to discover these biomarkers efficiently and precisely. We developed a scoring function system to identify RNA biomarkers with RNA-seq data for systems that have a limited number of cell types. We applied the method to two data sets, one for extracellular RNAs (ex-RNAs) and the other for intracellular microRNAs (miRNAs). The first data set has RNA-seq data of ex-RNAs from cell culture media for six different types of cells, including human embryonic stem cells. To get the RNA-seq data from intracellular miRNAs, we cultured three types of cells: human embryonic stem cells (H9), neural stem cells (NSC), hESC-derived endothelial cells (EC) and conducted small RNA-seq to their intracellular miRNAs. Using these data, we identified a set of ex-RNAs/smRNAs as candidates of biomarkers for different types of cells for cell manufacture. The validity of these findings was confirmed by the utilization of additional data sets and experimental procedures. We also used deep-learning-based prediction methods and simulated data to validate these discovered biomarkers.

Highly compliant biomimetic scaffolds for small diameter tissue-engineered vascular grafts (TEVGs) produced via melt electrowriting (MEW)

Biofabrication approaches toward the development of tissue-engineered vascular grafts (TEVGs) have been widely investigated. However, successful translation has been limited to large diameter applications, with small diameter grafts frequently failing due to poor mechanical performance, in particular mismatched radial compliance. Herein, melt electrowriting (MEW) of poly(ϵ-caprolactone) has enabled the manufacture of highly porous, biocompatible microfibre scaffolds with physiological anisotropic mechanical properties, as substrates for the biofabrication of small diameter TEVGs. Highly reproducible scaffolds with internal diameter of 4.0 mm were designed with 500 and 250 µm pore sizes, demonstrating minimal deviation of less than 4% from the intended architecture, with consistent fibre diameter of 15 ± 2 µm across groups. Scaffolds were designed with straight or sinusoidal circumferential microfibre architecture respectively, to investigate the influence of biomimetic fibre straightening on radial compliance. The results demonstrate that scaffolds with wave-like circumferential microfibre laydown patterns mimicking the architectural arrangement of collagen fibres in arteries, exhibit physiological compliance (12.9 ± 0.6% per 100 mmHg), while equivalent control geometries with straight fibres exhibit significantly reduced compliance (5.5 ± 0.1% per 100 mmHg). Further mechanical characterisation revealed the sinusoidal scaffolds designed with 250 µm pores exhibited physiologically relevant burst pressures of 1078 ± 236 mmHg, compared to 631 ± 105 mmHg for corresponding 500 µm controls. Similar trends were observed for strength and failure, indicating enhanced mechanical performance of scaffolds with reduced pore spacing. Preliminary in vitro culture of human mesenchymal stem cells validated the MEW scaffolds as suitable substrates for cellular growth and proliferation, with high cell viability (>90%) and coverage (>85%), with subsequent seeding of vascular endothelial cells indicating successful attachment and preliminary endothelialisation of tissue-cultured constructs. These findings support further investigation into long-term tissue culture methodologies for enhanced production of vascular extracellular matrix components, toward the development of the next generation of small diameter TEVGs.

Automated human induced pluripotent stem cell culture and sample preparation for 3D live-cell microscopy.

To produce abundant cell culture samples to generate large, standardized image datasets of human induced pluripotent stem (hiPS) cells, we developed an automated workflow on a Hamilton STAR liquid handler system. This was developed specifically for culturing hiPS cell lines expressing fluorescently tagged proteins, which we have used to study the principles by which cells establish and maintain robust dynamic localization of cellular structures. This protocol includes all details for the maintenance, passage and seeding of cells, as well as Matrigel coating of 6-well plastic plates and 96-well optical-grade, glass plates. We also developed an automated image-based hiPS cell colony segmentation and feature extraction pipeline to streamline the process of predicting cell count and selecting wells with consistent morphology for high-resolution three-dimensional (3D) microscopy. The imaging samples produced with this protocol have been used to study the integrated intracellular organization and cell-to-cell variability of hiPS cells to train and develop deep learning-based label-free predictions from transmitted-light microscopy images and to develop deep learning-based generative models of single-cell organization. This protocol requires some experience with robotic equipment. However, we provide details and source code to facilitate implementation by biologists less experienced with robotics. The protocol is completed in less than 10 h with minimal human interaction. Overall, automation of our cell culture procedures increased our imaging samples' standardization, reproducibility, scalability and consistency. It also reduced the need for stringent culturist training and eliminated culturist-to-culturist variability, both of which were previous pain points of our original manual pipeline workflow.

Plate-curving cell culture as a model for assessing curvature-induced cellular responses during neural tube morphogenesis

ABSTRACT Neurulation is an important shape-transforming event during embryonic development where a flat neural plate is converted into a neural tube. Failure in this morphogenetic process accounts for one of the most common birth defects. Mechanical biology has provided key insights into neural tube formation and curvature among many physical properties that are eliciting attention. However, the lack of a proper model to study the effect of curvature has limited the potential to reveal its role in neurulation. In this study, we introduce a novel cell culture method called plate-curving cell culture where a polydimethylsiloxane (PDMS) plate of desired physical properties is curved in either a concave or convex form while the human pluripotent stem cell culture induced to have early neural plate identity is placed on top of its surface. With this method, we observed the elongation of cell colony morphology, as well as the perpendicular alignment of the cell division axis in the concave surface; the oriented cell division does not seem to explain the colony elongation. Transcriptome comparison in search of alternate possibilities suggested selectively altered pathways in the concave surface culture. Our new method is widely available, easy-to-use and culture-friendly, facilitating future mechanobiological studies of neurulation.

Using a developed co-culture device to evaluate the proliferation of bone marrow stem cells by stimulation with platelet-rich plasma and electromagnetic field

BackgroundsBone marrow stem cell can differentiate to osteoblast by growth factors, pulsed low-intensity ultrasound and electric magnetic field. In the research, bone marrow stem cells were cultured; bone marrow stem cells in culture can be stimulated by platelet-rich plasma and electric field.MethodsThe culture well of the co-cultivation device has a radius of 7.5 mm and a depth of 7 mm. It is divided into two sub-chambers separated by a 3 mm high and 1 mm wide barrier. The bone marrow stem cells were seeded at a density of 2 × 104 cells and the medium volume was 120μl. Platelet-rich plasma (PRP) or platelet-poor plasma (PPP) was added to the other sub-chamber at a volume of 10μl. The bone marrow stem cells were subjected to different electric fields (0 ~ 1 V/cm) at a frequency of 70 kHz for 60 min.ResultsThe highest osteogenic capacity of bone marrow stem cells was achieved by addition of PRP to electric field stimulation (0.25 V/cm) resulted in a proliferation rate of 599.78%. In electric field stimulation (0.75 V/cm) with PPP, the proliferation rate was only 10.46%.ConclusionsBone marrow stem cell with PRP in the co-culture device combined with electric field at 0.25 V/cm strength significantly promoted the growth of bone marrow stem cells.

Enhancing wettability and adhesive properties of PVDF-based substrates through non-thermal helium plasma surface modification

Polyvinylidene fluoride (PVDF) has gained attention as a promising material for tissue engineering due to its biocompatibility and piezoelectricity. However, achieving proper cell adhesion to PVDF surfaces remains a challenge. This study focuses on the preparation and characterization of PVDF-based substrates, including PVDF thin films and nanocomposites incorporating CoFe2O4 magnetic nanoparticles. Helium plasma treatment is employed to modify the surface properties of these substrates. The plasma treatment induces significant changes in the topography of the PVDF-based nanocomposites. The roughness values of the samples increase from 2–3 nm to 14–17 nm after 90 s of plasma treatment, leading to enhanced hydrophilicity with an average contact angle below 60°. Importantly, the helium plasma treatment preserves the magnetic and structural properties of the substrates, suggesting the retention of their magnetoelectric properties and, thus treated composites hold potential for remote stem cell activation. We provide evidence of cytocompatibility and improved adhesive properties of plasma-treated substrates using human mesenchymal stem cell cultures.

The Influence of Exosomes Derived from Mesenchymal Stem Cells on the Development of Fibrosis In Vitro.

We studied the effect of exosomes derived from mesenchymal stem cells on the synthesis of collagen I and α-smooth muscle actin (α-SMA) by rat fibroblast culture. Exosomes were isolated from the verified culture of mesenchymal stem cells and also verified. Fibrosis was modeled using a fibroblast culture supplemented with recombinant TGF-β1 (5 ng/ml) and immunocytochemical analysis of the expression of collagen I and α-SMA markers was carried out. After 6-day incubation, the expression of the studied markers increased in comparison with the control. Addition of exosomes to the fibroblast culture reduced the production of collagen and SMA, which allows considering exosomes as a promising drug for the treatment of pathologies associated with fibrosis.

Isolation, Identification and Cultivation of Mesenchymal Stem Cells Derived from Adipose Tissue of Laying Hens

Isolation, Identification and Cultivation of Mesenchymal Stem Cells Derived from Adipose Tissue of Laying Hens

Abstract C035: Impact of South Florida environmental chemicals on breast cells derived from women of different ancestries

Abstract Breast Cancer (BC) occurrence is due to an interplay of ancestry-based and environmental factors. In order to study BC disparities in South Florida, our group initially focused on 7 Broward County zip codes where the proportion of BC identified at late stage is much higher (55%) than the national average (30-35%), and where there is a high percentage of people of African Ancestry (AA). Thus, we are investigating the interaction of ancestry and the environment in BC. Our hypothesis is that there are unique environmental sensitivities leading to BC that are associated with specific ancestral groups. We expect that some of the carcinogenic chemicals that cause BC may cause more advanced or aggressive BC in AA women. We are studying 2 environmental pollutants, one in water (arsenic) and one in soil (glyphosate) that are prevalent in S. Florida, and these local zip codes, and analyze the specific impact of these pollutants on unique breast reduction mammoplasty cultures derived from women of AA vs. Non-Hispanic White (NHW) ancestry. We have developed a unique tissue engineering system for non-diseased breast that allows us to establish cultures with high success (95%) using a stem cell-based medium and Matrigel. From primary cultures, we have established 38 extended explants (<13 passages) or cell lines (>13 passages). 10 are from AA women and 28 are from women of NHW ancestry, thus far with Hispanic samples in the works. We hypothesize that these groups will show significant differences in their gene expression patterns after exposure to environmental carcinogens. We have previously shown that there is a significantly higher proportion of breast stem cells in AA cultures (N=5) compared with the NHW cultures (N=5) (p= 0.028). This difference in size of the stem cell compartment may help to explain why the age of breast development in these two groups is significantly different and why their subtypes of BC are different based on ancestry. We are using 5 AA and 5 NHW breast reduction mammoplasty cultures, as well as stem cells derived from them, to test arsenic and glyphosate for their ability to alter the transcriptome of these cells using RNA sequencing. Analyses of gene expression using both supervised and unsupervised strategies will be performed (KEGG Pathways and single genes). We expect to see changes that are consistent with pre-invasive malignancy that have been established in endocrine disruptor studies. Because our group is one of the few that has successfully cultured stage 0 BC (Ductal Carcinoma In Situ), we have existing databases of transcriptomic changes from 6 DCIS cell lines as well as from 6 stage I BC cell lines for comparison. Preliminary data from the Nanostring BC360 and RNA sequencing show 8 genes involved in DNA damage and repair that are downregulated in AA breast reduction mammoplasty explants relative to NHW explants. This study will fill an important void in our understanding of how the environment affects pre-cancerous development in specific demographic populations. Citation Format: Jean J. Latimer, Yousef N. Alharbi, Colton R. Simmons, Stephen E. Zimberg, Essie K. Yates. Impact of South Florida environmental chemicals on breast cells derived from women of different ancestries [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C035.

Whole-Genome CRISPR-Cas9 Screen Identifies ZBTB7A As a Potential Therapeutic Target for Cda-II

Congenital dyserythropoietic anemia type II (CDA-II) is an autosomal recessive disease characterized by anemia, ineffective erythropoiesis, and increased bone marrow bi-nucleated erythroblasts. CDA-II is caused by loss-of-function mutations in SEC23B, which encodes a component of coat protein complex II (COP-II) vesicles/tubules that transport secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Mammals express two SEC23 paralogs, SEC23A and SEC23B. We have previously shown that SEC23A is functionally interchangeable with SEC23B and that increased expression of SEC23A rescues the SEC23B-deficient erythroid differentiation defect observed in CDA-II. Here, we utilized our recently generated clonal SEC23B deficient HUDEP-2 cell line to identify novel therapeutic targets for CDAII. SEC23B deficient HUDEP2 cells survive and grow normally when cultured as pro-erythroblasts in ‘maintenance media’. However, upon culturing these cells in ‘differentiation media’, which results in semi-synchronous erythroid differentiation, differentiated SEC23B-null HUDEP2 cells exhibit reduced growth, impaired differentiation, increased bi-nucleated erythroid cells, and erythroid cell death, features of CDA-II. To identify novel genes that play important roles in the pathophysiology of CDAII, we developed a functional genome-scale CRISPR knockout screen to define genes that when deleted, rescue the SEC23B-null erythroid differentiation defect. SEC23B null HUDEP2 cells cultured in maintenance media were infected with the hGeCKO-v2 lentiviral library at a multiplicity of infection (MOI) of ~0.3, allowing most transduced cells to receive 1 sgRNA (and Cas9) to knockout 1 gene only. Transduced cells were puromycin selected and passaged in expansion media for 14 days. SEC23B-null HUDEP2 cells were then differentiated for 10 days. Cells were harvested prior to differentiation (Day 0, D0), and differentiated (orthochromatic) cells were sorted at D10 of differentiation. Integrated sgRNAs were quantified by deep sequencing. sgRNAs targeting ZBTB7A were amongst the most enriched in D10 compared to D0 erythroid cells. To validate these findings, we transduced SEC23B-null HUDEP2 cells with two independent and efficient ZBTB7A-targeting sgRNAs. We found that ZBTB7A deletion (using either of the 2 sgRNAs) rescued the lethality and differentiation defect of SEC23B null HUDEP2 cells undergoing differentiation. We next generated 7 clonal cell lines with combined deletion for ZBTB7A and SEC23B and confirmed that these cells exhibited normal growth and differentiation indistinguishable from wildtype HUDEP2 cells. The rescuing effect of targeting ZBTB7A was also validated in SEC23B mutated human CD34+ hematopoietic stem and progenitor cell culture. Since SEC23A can functionally replace SEC23B in erythroid cells, we quantified the SEC23A mRNA level in SEC23B-null HUDEP2 cells deleted for ZBTB7A, by qRT-PCR. In early preliminary results, deletion of ZBTB7A resulted in a profound (~10 fold) increase in SEC23A mRNA expression, to a level predicted to be sufficient to rescue the SEC23B null erythroid differentiation defect. ChIP-seq analysis demonstrated that ZBTB7A occupies the SEC23A promoter in HUDEP2 cells. Taken together, these data suggest that ZBTB7A represses SEC23A expression during erythroid maturation and that in the setting of ZBTB7A deletion, SEC23A expression is de-repressed, resulting in rescue of the CDA-II erythroid differentiation defect. Genome editing of the ZBTB7A binding sites in the SEC23A promoter is ongoing in SEC23B-null HUDEP-2 cells. We will determine the impact of the latter editing on SEC23A expression and erythroid differentiation.

Adsorption of l-lactate from cell culture media by layered double hydroxide and evaluation of its cytotoxicity to cell lines

The large-scale culture of human induced pluripotent stem cells (hiPSCs) is essential for developing new pharmaceuticals and regenerative therapy methods. While for the development of cultured meat products, mass production of animal myoblasts is necessary. Both hiPSCs and animal myoblasts consume d-glucose as their energy source and produce l-lactate, which accumulates in cell culture media and inhibits cell proliferation. To make large-scale cell culture economically feasible, l-lactate removal and subsequent reuse of media are of high importance. The adsorption technique is attractive for l-lactate removal due to its low cost, ease of operation, and scalability. The current study is dedicated to 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES) intercalated Mg–Al layered double hydroxide (LDH), which acts as a biocompatible anion-exchanger in media. HEPES‧Mg–Al LDH was able to remove l-lactate from hiPS cells and myoblast-relevant media selectively while mostly retaining d-glucose. Adsorbent exhibited dose-dependent cytotoxicity to hiPSCs and C2C12 cells, mainly related to elevated osmolarity, HEPES, and Mg levels and adsorption of media micro components. By employing alternatively prepared sol-gel derived HEPES‧Mg–Al LDH, the required adsorbent dose for efficient l-lactate removal was reduced to a safe level. The current study thoroughly evaluates Mg–Al layered double hydroxides as suitable adsorbents for cell culture media regeneration and discusses the limitations of Mg–Al LDHs in systems relevant to hiPS cells and C2C12 cells. This work promotes the cost-effective large-scale production of cells and gives insight into the limitations of Mg–Al LDHs applied to systems of biological origin.

Editorial: Three-dimensional/3D stem cell culture systems

Editorial: Three-dimensional/3D stem cell culture systems

Mesenchymal Stem Cell in 3D Culture: Diminishing Cell Senescence in Cryopreservation and Long-term Expansion

Mesenchymal stem cells (MSCs) are widely recognized in cell treatment due to their capacity to secrete trophic factors, differentiate multipotent, and self-renew. Although there is growing evidence that MSCs have therapeutic benefits in various clinical settings, these cells eventually lose their ability to regenerate as they age, which increases cellular dysfunction. Several factors may affect MSCs aging, such as culture dimensions, cryopreservation process, and long-term expansion. Traditional two-dimensional (2D) culture conditions lack the complexities required to recreate MSCs in their natural environment. Meanwhile, three-dimensional (3D) culture mimics the niche, dynamic, and specialized microenvironments of the cells in vivo. The most used storage technique for MSCs, cryopreservation, requires a very low temperature reduction, which stresses cells and can cause the release of pro-inflammatory cytokines. For the utilization of MSCs in therapeutic applications, an in vitro expansion technique is required. Repeated expansion may reduce proliferative capacity, disrupts cellular shape, and impairs the somatic cell function of MSCs. Various processes and techniques may influence MSCs leading to cell aging. One of the culture methods, 3D culture, is shown to reduce the factors that will compromise the therapeutic effects of MSCs, especially cell senescence. The effect of culture dimensions, cryopreservation, and long-term expansion on cell senescence will be discussed in this review article.Keywords: cell aging, mesenchymal stem cell, 3D culture, cell senescence, cryopreservation, long-term expansion