Abstract Background Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) hold great therapeutic potential in regenerative medicine. Therefore, it is crucial to establish a Good Manufacturing Practice (GMP)-compliant methodology for the isolation and culture of WJ-MSCs. Through comprehensive research, encompassing laboratory-scale experiments to pilot-scale studies, we aimed to develop standardized protocols ensuring the high yield and quality of WJ-MSCs manufacturing. Methods Firstly, optimization of parameters for the enzymatic digestion method used to isolate WJ-MSCs was conducted. These parameters included enzyme concentrations, digestion times, seeding densities, and culture media. Additionally, a comparative analysis between the explant method and the enzymatic digestion method was performed. Subsequently, the consecutive passaging of WJ-MSCs, specifically up to passage 9, was evaluated using the optimized method. Finally, manufacturing processes were developed and scaled up, starting from laboratory-scale flask-based production and progressing to pilot-scale cell factory-based production. Furthermore, a stability study was carried out to assess the storage and use of drug products (DPs). Results The optimal parameters for the enzymatic digestion method were a concentration of 0.4 PZ U/mL Collagenase NB6 and a digestion time of 3 h, resulting in a higher yield of P0 WJ-MSCs. In addition, a positive correlation between the weight of umbilical cord tissue and the quantities of P0 WJ-MSCs has been observed. Evaluation of different concentrations of human platelet lysate revealed that 2% and 5% concentrations resulted in similar levels of cell expansion. Comparative analysis revealed that the enzymatic digestion method exhibited faster outgrowth of WJ-MSCs compared to the explant method during the initial passage. Passages 2 to 5 exhibited higher viability and proliferation ability throughout consecutive passaging. Moreover, scalable manufacturing processes from the laboratory scale to the pilot scale were successfully developed, ensuring the production of high-quality WJ-MSCs. Multiple freeze-thaw cycles of the DPs led to reduced cell viability and viable cell concentration. Subsequent thawing and dilution of the DPs resulted in a significant decrease in both metrics, especially when stored at 20–27 °C. Conclusion This study offers valuable insights into optimizing the isolation and culture of WJ-MSCs. Our scalable manufacturing processes facilitate the large-scale production of high-quality WJ-MSCs. These findings contribute to the advancement of WJ-MSCs-based therapies in regenerative medicine.

目的 验证基于吖啶橙(AO)/碘化丙啶(PI)荧光染色原理的自动细胞分析仪检测细胞因子诱导的杀伤细胞(CIK)数量及活率的可行性,并对CIK细胞培养全过程及冻存复苏后至使用前过程进行检测,建立相应的数量及活率标准.方法 采集健康供者的外周血分离、培养CIK细胞,取培养过程中的细胞使用AO/PI荧光染色法、应用自动细胞分析仪检测细胞数量及活率,对结果的专属性、准确度、精密度、数量线性及范围、活率线性及范围进行验证.应用建立的方法对外周血分离后的外周血单个核细胞(PBMC)、培养过程中、冻存前及复苏后的CIK细胞数量及活率进行全过程检测.结果 专属性、准确度、精密度、数量线性及范围、活率线性及范围验证均符合预期要求.CIK细胞培养全过程活率均不低于80％,样本平均倍增时间为(42.26±1.17)h.冻存前的CIK细胞在加入含5％DMSO的冷冻保护剂后90min内活率稳定,复苏后未经稀释或洗涤处理120min内活率稳定,复苏后经洗涤去除DMSO处理后12 h内活率稳定.结论 基于AO/PI荧光染色原理的自动细胞分析仪可以用于检测CIK细胞的数量及活率,其结果可以用于细胞放行评价及稳定性研究.

OBJECTIVE:To explore and design a novel bi-specific chimeric antigen receptor (CAR) structure. To obtain the corresponding CAR-T cells and verify killing effects on tumor cells in vitro and in vivo.METHODS:Five kinds of bi-specific CAR structures including humanized CD19 scFv and CD79b scFv, CD8 hinge & TM-4-1BB-CD3ζ and/or CD3ε chain intracellular regions were constructed and prepared. CAR-19-79b cells were obtained. Five kinds of CAR-T cells were co-incubated with the 3M-CD19-CD79b-Luc target cells. Luciferase assay and ELISA were used to detecte the killing ability of these five groups of CAR-T cells and the secretion of cytokines and compared. The optimal structure of CAR-T cells was used to treat the leukemia mouse model constructed by Daudi-Luc cells. And the treatment efficacy was evaluated. At the same time, other targets were used in this structure. With the same methods, the stability and effectiveness of the structure were verified.RESULTS:CAR-19-79b-T cells were cultured for 7 days, the expression rates of CAR-19 and CAR-79b were 21.6%-36.3% and 21.7%-37.8%, respectively. The killing rates of 5 kinds of CAR-19-79b-T cells prepared by T cells from 3 healthy donors on 3M-CD19-CD79b-Luc cells were significantly higher than those of the T cell control group at the effect-target ratio of 10∶1. Among them, the killing rates of CAR-19-79b-T cells with No. III and No. IV structures were the strongest. After co-incubation with 3M-CD19-CD79b-Luc target cells, the amount of IFN-γ and TNF-α secreted by CAR-T cells with CAR IV and CARV structures was the lowest. And there was no significance between the two groups (P>0.05). CAR IV cells with remarkable killing effect and low secretion factor had obvious therapeutic effect on Daudi-Luc leukemia mice, extending the survival period of mice to 64 days. And all mice in the T cell control group died at 41.0±2.4 days. The CAR-19-BCMA-T and CAR-19-22-T with the same structure showed significant killing ability and low cytokine expression levels.CONCLUSION:A novel bi-specific CAR structures was successfully designed, which could efficiently kill the corresponding tumor cells and secrete less cytokines (such as TNF-α, IFN-γ). Moreover, it shows obvious therapeutic effect on Daudi lymphoma mouse model. The bi-specific CAR structure shows good killing specificity and safety.

Background： Pulmonary fibrosis (PF) is a growing clinical problem with limited therapeutic options. Human umbilical cord mesenchymal stromal cell (hucMSC) therapy is being investigated in clinical trials for the treatment of PF patients. However, little is known about the underlying molecular and cellular mechanisms of hucMSC therapy on PF. In this study, the molecular and cellular behavior of hucMSC was investigated in a bleomycin induced mouse PF model. Methods: The effect of hucMSC on mouse lung regeneration was determined by detecting Ki67 expression and Edu incorporation in alveolar type 2 (AT2) and lung fibroblast cells. The hucMSC was transfected to express the membrane localized GFP before transplant into the mouse lung. The cellular behavior of hucMSC in mouse lung was tracked by GFP staining. Single cell RNA sequencing was performed to investigate the molecular mechanism of hucMSC on PF. Results: hucMSCs could alleviate collagen accumulation in lung and decrease the mortality of mouse induced by bleomycin. hucMSCs transplantation promoted AT2 cell proliferation and inhibited lung fibroblast cell proliferation. Mouse lung macrophage phenotype was changed after interacting with hucMSCs. By using single-cell RNA sequencing, a subcluster of interferon-sensitive macrophages were identified after hucMSC infusion. These macrophages elevate the secretion of CXCL9 and CXCL10 following hucMSC infusion and recruit more Treg cells to the injured lung. Conclusions: Our study establishes a link between hucMSCs, macrophage proliferation, and PF with potential applications in PF therapeutics. It provides new insights into how hucMSCs interact with macrophage during the repair process of bleomycin-induced PF and play its immunoregulation function.

Objective:To explore the immunosuppressive ability of human umbilical cord mesenchymal stem cells (hUCMSCs) on T and B lymphocytes before and after cryopreservation and provide future for using the clinical application of hUCMSCs.Methods:Human peripheral blood mononuclear cells were isolated from healthy human peripheral blood. Anti-CD3 and anti-CD8 monoclonal antibodies were used to activate T lymphocytes in vitro. Selected B lymphocytes were stimulated by ODN2395, human CD40 ligand, goat anti-human IgM antibodies and interleukin 2 in vitro. The cells were co-cultured with activated T and B lymphocytes, divided into a single stimulation group and hUCMSCs co-culture group (before and after cryopreservation) . The immunosuppressive ability of hUCMSCs on activated T and B lymphocytes were analyzed. In addition, the differences immunosuppressive ability of hUCMSCs before and after cryopreservation were also studied. The differences between groups were compared by t-test, and the differences among groups were compared by repeated measures design analysis of variance.Results:Compared with hUCMSCs before cypropreservation, the proliferation of CD3+T cells (37.60﹪±0.54﹪vs 39.40﹪±1.57﹪) , CD4+T cells (36.87﹪±0.54﹪vs 38.63﹪±1.39﹪) and CD8+T cells (40.37﹪±1.14﹪vs 42.47﹪±1.90﹪) (P > 0.05) were not significantly inhibited after cryopreservation. The apoptosis of CD3+T cells (18.07﹪±0.66﹪vs 16.77﹪±1.15﹪) , CD4+T cells (26.47﹪±1.13﹪vs 24.60﹪±1.47﹪) and CD8+T cells (3.52﹪±0.22﹪vs 2.72﹪±0.06﹪) had no significant difference (P > 0.05) . The proportion of regulatory cells (Treg) subsets (5.51﹪±0.71﹪vs 6.87﹪± 0.27﹪) had also no significant difference (P > 0.05) . hUCMSCs did not reduce the proportion of helper T cell (Th) subsets, especially the proportion of Th1 subsets (0.47﹪± 0.09﹪vs 0.33﹪± 0.04﹪) and Th17 subsets (0.21﹪± 0.04﹪vs 0.22﹪± 0.03﹪) (P > 0.05) . For B lymphocytes, hUCMSCs reduced the proportion of Th2 cell subsets (0.73﹪±0.07﹪vs 0.49﹪±0.06﹪) and IgM antibody secretion [ (739.70±28.39) ng/mL vs (560.21±3.81) ng/mL] after cryopreservation (P < 0.05) . There was no significant difference in proliferation and apoptosis of B lymphocytes, and reduced secretion of IgA and IgG antibodies before and after hUCMSCs cytopreservation (P > 0.05) .Conclusion:After cryopreservation, hUCMSCs were more powerful than those before cryopreservation in reducing the proportion of Th2 cell subsets and the secretion of IgM antibodies. However, the immunosuppressive abilities was same. Both hUCMSCs before and after cryopreservation have high clinical application value.

Based on the perspective of sustainability transitions (ST) theory, the paper dissects the opera-tional mechanisms of protected space driving emerging technology industrialization from an external system perspective, which is on the basis of defining essential characteristics and func-tional attributes of protected space. Meanwhile, it structures a three-dimensional evaluation model of protected space from the perspective of internal mechanism by introducing the policy mixes method. Finally, taking the Chinese New Energy Vehicle (NEV) industry as the example and using the system dynamics modeling method, the paper designs different simulation schemes from three dimensions of horizontal evolution trend, three types of policy instrument change and policy mixes of protected space, to predict and analyze the operation effect of protected space. The results show that the Chinese NEV industry is in the stage of market niche entry, and corresponding measures should be taken to increase the market scale, form effective demand and scale effect, and promote the diffusion of market niche; the effect of policy mixes is better than that of the single policy instrument; specific policy mixes should be adopted to develop simultaneously from both the supply and demand sides to improve the industrialization level of NEV.

Peripheral blood mononuclear cells are widely used as source material for anticancer immunotherapies. The conventional cryopreservation method for peripheral blood mononuclear cells is time-consuming and expansive, which involves controlled rate freezing followed by storage in liquid nitrogen. Instead, the convenient uncontrolled rate freezing cryopreservation method had been reported successfully in peripheral blood hematopoietic stem cells and peripheral blood progenitor cells. Therefore, we hypothesized that uncontrolled rate freezing cooling method maybe also applied to peripheral blood mononuclear cells cryopreservation. In this study, we evaluated the performance of uncontrolled rate freezing and controlled rate freezing cooling methods through cell recovery rate, viability, differentiation potential into cytokine-induced killer cells and the cellular properties of the cultured cytokine-induced killer cells. The results showed similar post-thaw viability and recovery rate in both controlled rate freezing and uncontrolled rate freezing cryopreserved peripheral blood mononuclear cells. Importantly, the uncontrolled rate freezing cryopreserved peripheral blood mononuclear cells exhibited higher growth ratio and earlier cell clustering during ex-vivo cytokine-induced killer cell culture than the controlled rate freezing ones. These two groups of expanded cytokine-induced killer cells also exhibited similar effector cell subset ratio and tumoricidal activity. In general, the performance of cryopreserved peripheral blood mononuclear cells using uncontrolled rate freezing cooling method, with the commercial cryoprotective agent CellBanker 2, was equal or better than the controlled rate freezing method. Our study implied that the combined use of cryoprotective agent CellBanker 2 and uncontrolled rate freezing could be a convenient cryopreservation method for peripheral blood mononuclear cells.

Mesenchymal stem cells (MSC) are a popular candidate in cellular therapy for many diseases. MSCs are well known by their feature of self-renewal and their differentiation potential. NESTIN is a cytoskeletal protein expressed in MSC that functions directly in cell proliferation and differentiation. Here, we demonstrated that adding UltraGRO, a medium supplement, could maintain and partially recover the expression of NESTIN in human umbilical cord derived MSCs (UC-MSCs). Furthermore, the UC-MSCs cultured with UltraGRO showed a better immunomodulation ability in a colitis mouse model compared with those cultured in other types of media. This indicates that the use of novel culture medium benefits the maintenance of NESTIN expression and NESTIN may be one of the vital factors that regulates the performance of MSCs.

In this study, we compared three commercially available and two widely used CPAs for their ability of cryopreserving PBMCs. Similar survival (81.0%) and recovery rate (73.7%) were observed among cells using these five CPAs. However, all the cryopreserved PBMCs exhibited a significantly lower survival rate when compared with the fresh samples (94.3%). We further evaluated effector cell subpopulation and tumoricidal activity of PBMC-derived cytokine-induced killing (CIK) cells and natural killing (NK) cells. Similar and high survival (CIK: 88.6%; NK: 87.5%) and recovery (CIK: 99.5%; NK: 99.7%) rates were detected in CIK and NK cells prepared from cryopreserved PBMCs using the five CPAs. The CD3(+)CD56(+) effector percentage (27.3%) of cryopreserved PBMC-derived CIK cells using the five different CPAs and their tumoricidal activities on melanoma CHL-1 cells (45.7%) and bladder cancer cell line T-24 (44.7%) were similar but significantly lower than those of the fresh PBMC-derived controls (effector: 30.7%; CHL-1: 84.2%; T-24: 82.2%). Cryopreserved PBMC-derived NK cells also exhibited similar tumoricidal activities (CHL-1: 73.8%; T-24: 71.9%) but was significantly lower than that of the fresh control group. We were not able to identify a specific CPA that performed superior than others in PBMC cryopreservation.