Establishment of a simple and efficient platform for car-t cell generation and expansion: from lentiviral production to in vivo studies

ABSTRACT Introduction: Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19 + B-cell malignancies in numerous clinical trials. The CAR molecule, which recognizes cell-surface tumor-associated antigen independently of human leukocyte antigen (HLA), is composed by one or more signaling molecules to activate genetically modified T cells for killing, proliferation, and cytokine production. Objectives: In order to make this treatment available for a larger number of patients, we developed a simple and efficient platform to generate and expand CAR-T cells. Methods: Our approach is based on a lentiviral vector composed by a second-generation CAR that signals through a 41BB and CD3-ζ endodomain. Conclusions: In this work, we show a high-level production of the lentiviral vector, which was successfully used to generate CAR-T cells. The CAR-T cells produced were highly cytotoxic and specific against CD19+ cells in vitro and in vivo, being able to fully control disease progression in a xenograft B-cell lymphoma mouse model. Our work demonstrates the feasibility of producing CAR-T cells in an academic context and can serve as a paradigm for similar institutions. Nevertheless, the results presented may contribute favoring the translation of the research to the clinical practice.

Stem cell expansion and hepatic differentiation: Measurement of related indicators and detection methods / 中国组织工程研究

BACKGROUND: Stem cells have wide application prospects in tissue engineering, regenerative medicine, cell therapy and drug research. In recent years, the applied research, such as bioartificial liver, requires large-scale and high-quality stem cells by expansion, and final obtains hepatocytes (hepatocyte-like cells) by directional differentiation. Through this way, how to efficiently obtain the differentiated cells, with excellent consistency, powerful function and uniform expression of markers, are the key problems to be solved. OBJECTIVE: To review the detection indicators and detection methods related to the process of stem cell expansion and hepatic differentiation, summarize the indicators and methods that have been applied to online detection, discuss the limitations of some indicators and methods applied to online detection, and prospect the indicators and detection technologies expected to be applied to online detection in the future. METHODS: PubMed, Web of Science, Elsevier and CNKI databases were retrieved for the articles concerning stem cell expansion and hepatic differentiation published from 1990 to 2019. The literature related to stem cell expansion and hepatic differentiation was collected. The keywords were “stem cells; expansion; hepatic differentiation; monitoring; real-time online” in English and Chinese, respectively. A total of 93 articles were searched, and finally 53 eligible articles were selected and reviewed. RESULTS AND CONCLUSION: There are many detection indicators and detection methods for stem cell expansion and hepatic differentiation. Among them, the detection indicators of the expansion process are mainly related to the maintenance of pluripotency, metabolic activity, survival rate, and cancer transformation trend of stem cells. The process of hepatic differentiation varies with cell types, in which, the pluripotent stem cells and the mesodermal lineage adult pluripotent stem cells should differentiate into the endoderm-oriented cells and then the mature hepatocytes (hepatocyte-like cells), while the endodermal lineage adult pluripotent stem cells mainly composed of hepatic stem/progenitor cells can directly differentiate into mature hepatocytes (hepatocyte-like cells). The specific markers, survival rate and metabolic activity of cells in different stages of differentiation are the focus of detection. At present, although the detection methods based on large-scale biochemical detection and analysis equipment have high reliability, they face the problems of poor real-time performance, high cost and difficulties in integration and miniaturization. Future concerns are focused on the screening of key detection indicators, quantification of detection criteria and realization of automatic online detection during stem cell expansion and hepatic differentiation.

Histological Method to Study the Effect of Shear Stress on Cell Proliferation and Tissue Morphology in a Bioreactor

BACKGROUND: Tissue engineering represents a promising approach for the production of bone substitutes. The use of perfusion bioreactors for the culture of bone-forming cells on a three-dimensional porous scaffold resolves mass transport limitations and provides mechanical stimuli. Despite the recent and important development of bioreactors for tissue engineering, the underlying mechanisms leading to the production of bone substitutes remain poorly understood. METHODS: In order to study cell proliferation in a perfusion bioreactor, we propose a simplified experimental set-up using an impermeable scaffold model made of 2 mm diameter glass beads on which mechanosensitive cells, NIH-3T3 fibroblasts are cultured for up to 3 weeks under 10 mL/min culture medium flow. A methodology combining histological procedure, image analysis and analytical calculations allows the description and quantification of cell proliferation and tissue production in relation to the mean wall shear stress within the bioreactor. RESULTS: Results show a massive expansion of the cell phase after 3 weeks in bioreactor compared to static control. A scenario of cell proliferation within the three-dimensional bioreactor porosity over the 3 weeks of culture is proposed pointing out the essential role of the contact points between adjacent beads. Calculations indicate that the mean wall shear stress experienced by the cells changes with culture time, from about 50 mPa at the beginning of the experiment to about 100 mPa after 3 weeks. CONCLUSION: We anticipate that our results will help the development and calibration of predictive models, which rely on estimates and morphological description of cell proliferation under shear stress.

Optimization of Large-Scale Expansion and Cryopreservation of Human Natural Killer Cells for Anti-Tumor Therapy

Allogeneic natural killer (NK) cell therapy is a potential therapeutic approach for a variety of solid tumors. We established an expansion method for large-scale production of highly purified and functionally active NK cells, as well as a freezing medium for the expanded NK cells. In the present study, we assessed the effect of cryopreservation on the expanded NK cells in regards to viability, phenotype, and anti-tumor activity. NK cells were enormously expanded (about 15,000-fold expansion) with high viability and purity by stimulating CD³⁺ T cell-depleted peripheral blood mononuclear cells (PBMCs) with irradiated autologous PBMCs in the presence of IL-2 and OKT3 for 3 weeks. Cell viability was slightly reduced after freezing and thawing, but cytotoxicity and cytokine secretion were not significantly different. In a xenograft mouse model of hepatocellular carcinoma cells, cryopreserved NK cells had slightly lower anti-tumor efficacy than freshly expanded NK cells, but this was overcome by a 2-fold increased dose of cryopreserved NK cells. In vivo antibody-dependent cell cytotoxicity (ADCC) activity of cryopreserved NK cells was also demonstrated in a SCID mouse model injected with Raji cells with rituximab co-administration. Therefore, we demonstrated that expanded/frozen NK cells maintain viability, phenotype, and anti-tumor activity immediately after thawing, indicating that expanded/frozen NK cells can provide ‘ready-to-use’ cell therapy for cancer patients.

Generation and Characterization of Human Osteoarthritis Cartilage-derived Mesenchymal Stem Cells by Modified Sample Processing and Culture Method

@#Introduction: Mesenchymal stem cells (MSCs) can be isolated from different tissue sources, and show a high differentiation capacity towards osteogenic, adipogenic, chondrogenic, neurogenic and myogenic lineages upon a specific induction. Although the retrieval of MSCs from normal tissues is very straightforward, yet it could be challenging in degenerative conditions that limit the expansion of stem cells such as osteoarthritis. Thus, this study aimed to establish human MSCs culture from osteoarthritic cartilage (OA hC-MSCs) by optimising the sample processing and culture techniques. Methods: Human osteoarthritis knee cartilage samples were obtained (2-4 g) from 8 patients with a mean age of 62.75 years old during the joint replacement surgery. A conventional culture method carried along with the modified method where the period of enzyme digestion and serial plating culture procedure were incorporated. Results: The modified culture method has significantly increased the number of single cells twice after the sample processing. The time taken to form colonies and achieve confluence was also reduced when samples subjected to the modified method. The number of cell yields after passage 0 for the conventional and modified methods were 3.05±0.31 and 6.10±0.42 million cells, respectively. The adherent cells generated under these two conditions comply with criteria for MSCs in term of immunophenotyping and mesodermal differentiation. Conclusions: The current modified method enhances the production of MSCs and could be opted for samples that known to have reduced or defective stem cell pool which may impede the in vitro cell expansion.

Dynamic Changes of Mineral Element in The Cell Wall of Growth Cells Detected by CSEM-EDX / 生物化学与生物物理进展

Aerenchyrna formation has been described in depth in a number of species at a histological level. But large gaps remain in our understanding of its regulation as a developmental process. It is attempted to analyse essential mineral elements like K, Mg, Cu, Zn, Ca and P in the cell wall of aerenchyma cells in petioles ofS. trifolia at five different developmental stages by CSEM-EDX technique. At early stage, K and Cl concentrations in cell wall were high up to 36% and 4.3% of dry weight, respectively. It supported the hypotheses that aerenchyma spaces are filled with liquid at early developmental stages of aerenchyma in S. trifolia petiole. Mg concentration was high at stage 2, up to 0.86% of dry weight. Zinc and Cu were detected only at rapid expansion stages, during which the concentrations were up to 1.5% and 2.5%, respectively. Calcium was detected in the cell wall only at mature stages, the concentration was high up to 1.3% of dry weight at stages 4 and 5. These results confirmed that the element concentration of aerenehyma cell wall undergoes dynamic changes during different developmental stages, and a low Ca with high Zn and Cu concentration are needed for cell expansion. Copper and Zn deposition in the cell wall showed a significant positive linear correlation, suggesting that these two elements share same or similar uptake and transport mechanism in plants.

Apheresis: the Italian experience / Aférese: a experiência italiana

The Italian experience is a long one, beginning with granulocyte collection in the late Bruni R. months of 1972 and progressively expanding to new application and new techniques, many of which Italian in origin and diffusion. This is true for sequestration, multiple Carlier P. component collection, ascitapheresis, dy-platelet collection, but is also true for the new application of known techniques such as cascade, filtration for disorders such as acute Guillain Barr Syndrome, KT, Cyclosporin induced or secondary hypertriglericeridemia, lepromotous vasculitis leptospirosis, hyperacute kidney rejection, autoimmune pure redcell aplasia and many other disorders treated by plasma exchange for the first time in Italy and in general terms in Italy this intermediate level of complexity techniques have found a wide if not enthusiastic acceptance. Twenty-four years later the general appreciation and interest have not modified their impact onto transfusion medicine and Italy continues to be among the leaders also because of the presence in the country of a couple of industries of international excellence involved in apheresis and/or related fields. The presence of Italy in apheresis was marked by the first international Society (ESFH, European Society for Hemapheresis) set up in 1982, with its first meeting organized in Florence the following year and the one of 2001 that will be held in Italy. The Italian presence in apheresis is also marked by the large Italian participation in the international meetings, frequently as invited speakers and chairmen. Furthermore, many well recognized investigators got their training in Italy and Italy is proud of their achievements.

A experiência italiana em aféreses é antiga e teve início nos últimos meses de 1972 e progressivamente se expandiu com novas aplicações e novas técnicas, muitas delas de origem italiana. Isto é real para sequestração, coleção multipla de componentes, citaféreses e coleção de plaquetas, mas também é verdade que novas aplicações como na sindrome de Guillain-Barré, PTT, hipertrigliceridemia secundária a ciclosporina, vasculite lepromatosa, leptospirose, rejeição renal aguda.

The application of CD34-immunoaffinity column in the isolation and expansion of hematopoietic stem and progenitor cells of cord blood / 中国免疫学杂志

Objective:To observe the effect of CD34 immunoaffinity column on the isolation of hematopoietic stem and progenitor cells of cord blood and the proliferatory and expansion characteristics of CD34 + cells in vitro.Methods:CD34 +cells were isolated from cord blood using CD34 immunoaffinity column.Cell surface antigens were analysed by FACS.The separated and un separated cells were cultured with human hematopoietic growth factors(HGFS)in liquid culture system and CFU GEMM culture system.Results:CD34 + cells were enriched with a purity of 49.62%?17.69% and a recovery of 54.38%?11.91% using the immunoaffinity column.The separated CD34 +cells and cord blood MNC expanded to 561.00 folds and 44.44 folds respectively after cultured with HGFS for 20 days.The percentages of CD34 + cells in separated group and control were 53.38% and 7.91% respectively after cultured for 12 days.The number of CFU GEMM in separated group was significantly higher than that in control(P

The ex vivo multiplication of ??T cell from the patient with nasopharyngeal carcinoma and its expression of V? gene / 解放军医学杂志

80%). The resulting bulk cultures were mainly comprised of a CD56~- subset of ??T cells. The expression of V?1, V?2 and V?3 gene families in the ex vivo multiplied ??T cells from TIL and PBMC of patients with NPC, and PBMC of normal control was demonstrated by RT-PCR. Conclusion The ??TIL of NPC can be multiplied in vitro for the first time. The subsets (V?1, V?2 and V?3) of ??T cells from PBMC of healthy individuals, PBMC and TIL of patients with NPC, can also be multiplied in vitro, and the experiment lays an experimental foundation of using ??T cells for the cellular adoptive therapy in patients with NPC.