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Mesenchymal‐derived stromal or progenitor cells, commonly called “MSCs,” have attracted significant clinical interest for their remarkable abilities to promote tissue regeneration and reduce inflammation. Recent studies have shown that MSCs' therapeutic effects, originally attributed to the cells' direct differentiation capacity into the tissue of interest, are largely driven by the biomolecules the cells secrete, including cytokines, chemokines, growth factors, and extracellular vesicles containing miRNA. This secretome coordinates upregulation of endogenous repair and immunomodulation in the local microenvironment through crosstalk of MSCs with host tissue cells. Therapeutic applications for MSCs and their secretome‐derived products often involve in vitro monolayer expansion. However, consecutive passaging of MSCs significantly alters their therapeutic potential, inducing a broad shift from a pro‐regenerative to a pro‐inflammatory phenotype. A consistent by‐product of in vitro expansion of MSCs is the onset of replicative senescence, a state of cell arrest characterized by an increased release of proinflammatory cytokines and growth factors. However, little is known about changes in the secretome profile at different stages of in vitro expansion. Some culture conditions and bioprocessing techniques have shown promise in more effectively retaining the pro‐regenerative and anti‐inflammatory MSC phenotype throughout expansion. Understanding how in vitro expansion conditions influence the nature and function of MSCs, and their associated secretome, may provide key insights into the underlying mechanisms driving these alterations. Elucidating the dynamic and diverse changes in the MSC secretome at each stage of in vitro expansion is a critical next step in the development of standardized, safe, and effective MSC‐based therapies. Throughout in vitro expansion, MSCs and their secretome undergo a shift from a pro‐regenerative to a proinflammatory phenotype. Changes underlying this shift include alterations in DNA methylation profile, morphological abnormalities, telomere shortening, oxidative stress, senescence, and secreted biomolecules (cytokines, chemokines, growth factors, MSC‐EVs). Approaches that have shown promise in retaining the functional properties of culture‐expanded MSCs include cellular preconditioning (hypoxia, inflammatory cytokines, 3D culturing), pharmacological interventions (senolytics), and telomerase overexpression.

Natural killer cells constitute a part of the innate immune system that mediates an effective immune response towards virus-infected and malignant cells. In recent years, research has focused on exploring and advancing NK cells as an active immunotherapy platform. Despite major advances, there are several key challenges that need to be addressed for the effective translation of NK cell research to clinical applications. This review highlights some of these challenges and the innovative strategies being developed to overcome them, including in vitro expansion, in vivo persistence, infiltration to the tumor site, and prevention of exhaustion.

Chimeric antigen receptor (CAR)-T cells are engineered to identify and eliminate cells expressing a target antigen. Current manufacturing protocols vary between commercial CAR-T cell products warranting an assessment of these methods to determine which approach optimally balances successful manufacturing capacity and product efficacy. One difference between commercial product manufacturing methods is whether T cell engineering begins with fresh (unfrozen) patient cells or cells that have been cryopreserved prior to manufacture. Starting with frozen PBMC material allows for greater manufacturing flexibility, and the possibility of collecting and storing blood from patients prior to multiple lines of therapy. We prospectively analyzed if second generation anti-CD19 CAR-T cells with either CD28 or 4-1BB co-stimulatory domains have different phenotype or function when prepared side-by-side using fresh or cryopreserved PBMCs. We found that cryopreserved PBMC starting material is associated with slower CAR-T cell expansion during manufacture but does not affect phenotype. We also demonstrate that CAR-T cell activation, cytokine production and in vitro anti-tumor cytotoxicity were not different when CAR-T cells were manufactured from fresh or cryopreserved PBMC. As CAR-T cell therapy expands globally, the need for greater flexibility around the timing of manufacture will continue to grow. This study helps support the concept that cryopreservation of PBMCs could be the solution to these issues without compromising the quality of the final CAR-T product.

The purpose of this work is to describe the use of Fibrin-Plasma Rich in Growth Factors (PRGF) membranes for the treatment of a rabbit alkali-burn lesion. For this purpose, an alkali-burn lesion was induced in 15 rabbits. A week later, clinical events were evaluated and rabbits were divided into five treatment groups: rabbits treated with medical treatment, with a fibrin-PRGF membrane cultured with autologous or heterologous rabbit Limbal Epithelial Progenitor Cells (LEPCs), with a fibrin-PRGF membrane in a Simple Limbal Epithelial Transplantation and with a fibrin-PRGF membrane without cultured LEPCs. After 40 days of follow-up, corneas were subjected to histochemical examination and immunostaining against corneal or conjunctival markers. Seven days after alkali-burn lesion, it was observed that rabbits showed opaque cornea, new blood vessels across the limbus penetrating the cornea and epithelial defects. At the end of the follow-up period, an improvement of the clinical parameters analyzed was observed in transplanted rabbits. However, only rabbits transplanted with cultured LEPCs were positive for corneal markers. Otherwise, rabbits in the other three groups showed positive staining against conjunctival markers. In conclusion, fibrin-PRGF membrane improved the chemically induced lesions. Nonetheless, only fibrin-PRGF membranes cultured with rabbit LEPCs were able to restore the corneal surface.

Primordial germ cells (PGCs) are the founding population of the germ cell lineage that undergo a multistep process to generate spermatozoa or oocytes. Establishing an appropriate culture system for PGCs is a key challenge in reproductive biology. By a chemical screening using mouse PGC-like cells (mPGCLCs), which were induced from mouse embryonic stem cells, we reported previously that forskolin and rolipram synergistically enhanced the proliferation/survival of mPGCLCs with an average expansion rate of ∼20-fold. In the present study, we evaluated other chemicals or cytokines to see whether they would improve the current mPGCLC culture system. Among the chemicals and cytokines examined, in the presence of forskolin and rolipram, cyclosporin A (CsA) and fibroblast growth factors (FGFs: FGF2 and FGF10) effectively enhanced the expansion of mPGCLCs in vitro (∼50-fold on average). During the expansion by CsA or FGFs, mPGCLCs comprehensively erased their DNA methylation to acquire a profile equivalent to that of gonadal germ cells in vivo, while maintaining their highly motile phenotype as well as their transcriptional properties as sexually uncommitted PGCs. Importantly, these mPGCLCs robustly contributed to spermatogenesis and produced fertile offspring. Furthermore, mouse PGCs (mPGCs) cultured with CsA ex vivo showed transcriptomes and DNA methylomes similar to those of cultured mPGCLCs. The improved culture system for mPGCLCs/mPGCs would be instructive for addressing key questions in PGC biology, including the mechanisms for germ cell migration, epigenetic reprogramming, and sex determination of the germline. Summary sentence Cyclosporin A and fibroblast growth factors enhanced the proliferation/survival of mouse primordial germ cell-like cells in vitro with the retention of a capacity to contribute to spermatogenesis.

The methodologies and applications of ex vivo expansion of umbilical cord blood cells represent a significant research domain, primarily owing to the distinctive characteristics of these cells and their prospective uses in regenerative medicine and hematopoietic stem cell transplantation. We searched PubMed and Web of Science using the terms “umbilical cord blood cells,” “cytokine,” “haematopoietic cells,” “umbilical cord mesenchymal stem cells,” “in vitro expansion,” and “microenvironment,” and the selected literature was organized and analyzed. We present a narrative review of the mechanisms by which umbilical cord mesenchymal stem cells enhance the in vitro expansion of umbilical cord blood-derived hematopoietic stem cells, focusing on three primary aspects: secretion of diverse cytokines to replicate the natural hematopoietic microenvironment, transmission of critical signals via direct cell-to-cell contact, and exertion of immunomodulatory effects to alleviate environmental stress. Although these processes can significantly promote the proliferation and survival of hematopoietic stem cells, the challenge of concurrently preserving the long-term stemness of these cells in an in vitro environment remains a critical issue for future research.

Background The extended use of mesenchymal stromal cells (MSC) for cellular therapies raises safety concerns, and preliminary controls are needed before clinical application to guarantee patient safety. In vitro expansion is frequently required to obtain sufficient material, with an increased risk of appearance and selection of genetically altered or senescent cells, which could impair not only safety but also the biological potential and therapeutic efficacy of these cells. In vitro manipulated MSCs must adhere to the guidelines of Good Manufacturing Practices including the verification of genomic stability, transformation potential and senescence. Several tests are currently used, mainly karyotyping, which enables the detection of large alterations. Small alterations, albeit potentially important, are usually not evaluated, except for selected known mutations in a few crucial genes. Methods Here, the potential of Next Generation Sequencing (NGS) to detect small DNA alterations was exploited as a reliable tool to monitor the genetic stability of cells manipulated prior to therapeutic application. The acquisition of small somatic mutations during in vitro culture of human adipose-derived mesenchymal stromal cells from 23 osteoarthritis patients was investigated at different times from passage 1 to passage 10 using targeted NGS of a panel of human cancer-related genes. Moreover, copy number variations in selected genes were analyzed using both the ‘Coverage Analysis’ plugin and digital PCR. Results Overall genetic stability was maintained, despite the emergence of sporadic variants, some with pathogenic potential or of uncertain significance, reflecting dynamic changes in culture. No copy number variations occurred during in vitro culture. Conclusions Time in culture is confirmed as a key variable influencing the acquisition of genetic alterations, underscoring the importance of minimizing expansion times to preserve genetic stability. Given the potential impact of small genetic alterations on cellular function and the high next generation sequencing processivity and throughput, exploring incoming mutations in MSC cells via NGS turns out to be not only a perspective research pursuit, but also a robust strategy for surveillance of genetic stability. Integrating this approach into quality control guidelines would support standardized monitoring of the genomic integrity of manipulated MSCs prior to clinical application.

Given recent progress in regenerative medicine, we need a means to expand chondrocytes in quantity without losing their regenerative capability. Although many reports have shown that growth factor supplementation can have beneficial effects, the use of growth factor–supplemented basal media has widespread effect on the characteristics of chondrocytes. Chondrocytes were in vitro cultured in the 2 most widely used chondrocyte growth media, conventional chondrocyte culture medium and mesenchymal stem cell (MSC) culture medium, both with and without fibroblast growth factor-2 (FGF2) supplementation. Their expansion rates, expressions of extracellular matrix–related factors, senescence, and differentiation potentials were examined in vitro and in vivo. Our results revealed that chondrocytes quickly dedifferentiated during expansion in all tested media, as assessed by the loss of type II collagen expression. The 2 basal media (chondrocyte culture medium vs. MSC culture medium) were associated with distinct differences in cell senescence. Consistent with the literature, FGF2 was associated with accelerated dedifferentiation during expansion culture and superior redifferentiation upon induction. However, chondrocytes expanded in FGF2-containing conventional chondrocyte culture medium showed MSC-like features, as indicated by their ability to direct ectopic bone formation and cartilage formation. In contrast, chondrocytes cultured in FGF2-supplemented MSC culture medium showed potent chondrogenesis and almost no bone formation. The present findings show that the chosen basal medium can exert profound effects on the characteristics and activity of in vitro–expanded chondrocytes and indicate that right growth factor/medium combination can help chondrocytes retain a high-level chondrogenic potential without undergoing hypertrophic transition.

Satellite cells (SCs) are muscle stem cells capable of regenerating injured muscle. The study of their functional potential depends on the availability of methods for the isolation and expansion of pure SCs with preserved myogenic properties after serial passages in vitro. Here, we describe the ice-cold treatment (ICT) method, which is a simple, economical, and efficient method for the isolation and in vitro expansion of highly pure mouse and human SCs. It involves a brief (15–30 min) incubation on ice (0 °C) of a dish containing a heterogeneous mix of adherent muscle mononuclear cells, which leads to the detachment of only the SCs, and gives rise to cultures of superior purity compared to other commonly used isolation methods. The ICT method doubles up as a gentle passaging technique, allowing SC expansion over extended periods of time without compromising their proliferation and differentiation potential. Moreover, SCs isolated and expanded using the ICT method are capable of regenerating injured muscle in vivo. The ICT method involves minimal cell manipulation, does not require any expertise or expensive reagents, it is fast, and highly reproducible, and greatly reduces the number of animals or human biopsies required in order to obtain sufficient number of SCs. The cost-effectiveness, accessibility, and technical simplicity of this method, as well as its remarkable efficiency, will no doubt accelerate SC basic and translational research bringing their therapeutic use closer to the clinic.

Human mesenchymal stem cells (hMSCs) are widely used in regenerative medicine, but large-scale in vitro expansion alters their function, impacting proliferation and differentiation potential. Currently, a predictive marker to assess these changes is lacking. Here, we used dielectrophoresis (DEP) to characterize the electrical phenotype of hMSCs derived from bone marrow (BM), adipose tissue (AT), and umbilical cord (UC) as they aged in vitro from passage 4 (P4) to passage 9 (P9). The electrical phenotype was defined by the DEP spectra, membrane capacitance, and cytoplasm conductivity. Cell morphology and size, growth characteristics, adipogenic differentiation potential, and osteogenic differentiation potential were assessed alongside label-free biomarker membrane capacitance and cytoplasm conductivity. Differentiation was confirmed by histological staining and RT-qPCR. All hMSCs exhibited typical morphology, though cell size varied, with UC-hMSCs displaying the largest variability across all size metrics. Growth analysis revealed that UC-hMSCs proliferated the fastest. The electrical phenotype varied with cell source and in vitro age, with high passage hMSCs showing noticeable shifts in DEP spectra, membrane capacitance, and cytoplasm conductivity. Correlation analysis revealed that population doubling level (PDL) correlated with membrane capacitance and cytoplasm conductivity, indicating PDL as a more precise marker of in vitro aging than passage number. Additionally, we demonstrate that membrane capacitance correlates with the osteogenic marker COL1A1 and that cytoplasm conductivity correlates with the adipogenic markers ADIPOQ and FABP4, suggesting that DEP-derived electrical properties serve as label-free biomarkers of differentiation potential. While DEP has previously been applied to BM-hMSCs and AT-hMSCs, and more recently to UC-hMSCs, few studies have provided a direct comparison across all three sources or tracked changes across continuous expansion. These findings underscore the utility of DEP as a label-free approach for assessing hMSC aging and function, offering practical applications for optimizing stem cell expansion and stem cell banking in clinical settings.