RESUMO
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.
Assuntos
Células-Tronco Mesenquimais , Geleia de Wharton , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Humanos , Geleia de Wharton/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células Cultivadas , Proliferação de Células , Separação Celular/métodos , Separação Celular/normasRESUMO
Adipose-derived stem cells (ADSCs) have raised big interest in therapeutic applications in regenerative medicine and appear to fulfill the criteria for a successful cell therapy. Their low immunogenicity and their ability to self-renew, to differentiate into different tissue-specific progenitors, to migrate into damaged sites, and to act through autocrine and paracrine pathways have been altogether testified as the main mechanisms whereby cell repair and regeneration occur. The absence of standardization protocols in cell management within laboratories or facilities added to the new technologies improved at patient's bedside and the discrepancies in cell outcomes and engraftment increase the limitations on their widespread use by balancing their real benefit versus the patient safety and security. Also, comparisons across pooled patients are particularly difficult in the fact that multiple medical devices are used and there is absence of harmonized assessment assays despite meeting regulations agencies and efficient GMP protocols. Moreover, the emergence of the COVID-19 breakdown added to the complexity of implementing standardization. Cell- and tissue-based therapies are completely dependent on the biological manifestations and parameters associated to and induced by this virus where the scope is still unknown. The initial flow chart identified for stem cell therapies should be reformulated and updated to overcome patient infection and avoid significant variability, thus enabling more patient safety and therapeutic efficiency. The aim of this work is to highlight the major guidelines and differences in ADSC processing meeting the current good manufacturing practices (cGMP) and the cellular therapy-related policies. Specific insights on standardization of ADSCs proceeding at different check points are also presented as a setup for the cord blood and bone marrow.
Assuntos
Tecido Adiposo/citologia , COVID-19 , Separação Celular/normas , Transplante de Células-Tronco/normas , Células-Tronco/citologia , HumanosRESUMO
Peripheral blood from healthy sheep (n = 3) and goats (n = 3) were employed to establish an efficient method for simultaneous isolation of peripheral blood mononuclear cells (PBMCs) and neutrophils and to standardize protocols for monocyte purification and generation of monocyte-derived macrophages (MDMs). In both species, a significantly enriched population of PBMCs, with higher purity and number of cells determined by flow cytometry, was achieved when processing through a density gradient a mixture of buffy-coat and red blood cell layer (RBC) in comparison to the use of just the buffy-coat (p < 0.05). Neutrophils could be subsequently isolated from the layer, located underneath PBMCs fraction with significant higher purity rates, higher than 85 % determined by flow cytometry, than those obtained with protocols without density gradients (< 60 %) (p < 0.05). This technique would allow the isolation of both cell populations from the same sample of blood. A pure cell population of monocytes, CD14+ cells, was purified from PBMCs when using immunomagnetic columns, which allow for 17 % (nº monocytes/nº PBMCs) of yield and high percentages of expression of CD14+ (88 %), MHC-II+ (91.5 %) and CD11b+ (94 %) established by flow cytometry. On the other hand, the classical and non-expensive purification of monocytes from PBMCs based on the adherence capacity of the former, allowed significantly lower yield of monocytes (4.6 %), with percentages of surface markers expression that dropped to 35 %, 65 % and 55 %, respectively (p < 0.001), suggesting the isolation of a mixed population of cells. The addition of GM-CSF to the culture, at concentration from 25 to 125 ng/mL, enhanced proportionally the number of MDMs generated compared to the absence of supplementation or the use of autologous serum from 5% to 20 %. However, purification of monocytes through the adherence method achieved higher yields of MDMs than those isolated through immunomagnetic columns in both species (p < 0.001). Under the conditions of this study, the use of centrifugation in density gradients allow for the simultaneous purification of PBMCs and neutrophils, with high purity of both populations, from the same sample of blood. The isolation of monocytes could be subsequently achieved through two different methods, i.e. based on immunomagnetic columns or adherence. The preference between both methods would depend on the necessities of the experiment, the initial sample with high purity of monocytes or a final population of MDMs required.
Assuntos
Contagem de Células/métodos , Separação Celular/métodos , Separação Celular/normas , Leucócitos Mononucleares/fisiologia , Macrófagos/fisiologia , Ruminantes/imunologia , Animais , Contagem de Células/normas , Diferenciação Celular , Células Cultivadas/imunologia , Células Dendríticas/imunologia , Cabras/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Leucócitos/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Monócitos/imunologia , Ovinos/imunologiaRESUMO
OBJECTIVE: To evaluate whether specific ovarian decortication techniques vary in promoting ovarian cortex cryopreservation and transplant outcomes. DESIGN: Experimental design. SETTING: University hospital. ANIMAL(S): Nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) female mice. INTERVENTION(S): Human ovarian biopsy samples allocated to one of the following decortication procedures: scratching with scalpel blade (B), cutting with microsurgical scissors (M), separation with slicer (S), or no-separation (control, C). Parallel, in vivo experiment: decortication techniques combined with slow freezing (SF) and vitrification (VT) before xenograft into immunodeficient mice. MAIN OUTCOME MEASURE(S): Follicular counts, apoptosis, shear stress, Hippo pathway and inflammation. In vivo: recovered grafts analyzed for follicular counts, angiogenesis, proliferation, and fibrosis. RESULT(S): There were no differences in follicular density or number of damaged follicles between the decortication techniques in the in vitro study. Nevertheless, the M samples showed statistically significantly increased stromal damage compared with the controls and S samples, and up-regulation of Hsp60 shear stress gene expression. Decortication by both M and S inhibited the Hippo pathway, promoting gene expression changes. In the 21-day xenograft, total follicular density statistically significantly decreased compared with the nongrafted controls in all groups. Nevertheless, no differences were observed between the decortication techniques. Ovarian stroma vascularization was increased in the vitrified samples, but among the slow-freezing samples, the B samples had the lowest microvessel density. The M decorticated xenografts had increased fibrosis. CONCLUSION(S): Decortication with a slicer causes less damage to ovarian tissue than other commonly used methods although microsurgical scissors seem to preserve slightly increased follicular numbers. Nevertheless, blade decortication seems to be a reliable technique for maintaining acceptable follicular conditions without inducing serious stromal impairment.
Assuntos
Separação Celular/normas , Criopreservação/normas , Folículo Ovariano/fisiologia , Ovário , Células Estromais/citologia , Coleta de Tecidos e Órgãos/normas , Adolescente , Adulto , Animais , Calibragem , Separação Celular/métodos , Sobrevivência Celular , Criopreservação/métodos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Folículo Ovariano/citologia , Controle de Qualidade , Coleta de Tecidos e Órgãos/métodos , Adulto JovemRESUMO
BACKGROUND: Recent advances in therapeutic interventions have dramatically improved complete response rates in patients with multiple myeloma (MM). The ability to identify residual myeloma cells (e.g., measurable residual disease [MRD]) can provide valuable information pertaining to patient's depth of response to therapy and risk of relapse. Multiparametric flow cytometry is an excellent technique to monitor MRD and has been demonstrated to correlate with patient outcome post-treatment. To achieve the high sensitivity (one abnormal cell in 105 -106 cells) required for MRD evaluation, millions of cells have to be acquired and conventional immunophenotyping protocols are unable to attain these numbers, indicating the needs for alternative flow cytometric staining procedures. A bulk, "Pre-lysis" method is the consensus approach for staining large number of cells, requires two red blood cell lysis steps, and can adversely affect epitope density. In this study, we tested the "Pooled-tube" and "Dextran Sedimentation" staining procedures and correlated them with the "Pre-lysis" method as potential alternative approaches. METHODS: A total of 22 bone marrow aspirates from patients with plasma cell (PC) dyscrasia were processed in parallel using the "Pre-lysis," "Pooled-tube," and "Dextran Sedimentation" techniques. Stain indices were calculated and compared to assess their impacts on staining performance for each antibody used in the consensus panel. The recovery of normal and abnormal PCs, mast cells, and B cell precursors was enumerated and compared after their counts were normalized using fluorescent beads. The limit of blank, limit of detection, and lower limit of quantification were established using serial dilution experiments. RESULTS: The staining performances of CD19 PECy7, CD27 BV510, CD81 APCH7, and CD138 BV421 were improved using the "Pooled-tube" method when compared to "Pre-lysis." "Pre-lysis" was better at resolving CD56 using clone C5.9 but our results demonstrated similar improvement can also be achieved by "Pooled-tube" when alternative CD56 PE clones were used. "Dextran sedimentation" yielded similar staining results when compared to "Pre-lysis" for all the markers analyzed. The "Pooled-tube" method, when normalized to "Pre-lysis," recovered higher numbers of total PCs (1.2 ± 0.2 times higher; p = .049), normal PCs (1.4 ± 0.26; p = .007), mast cells (1.46 ± 0.27; p = .003), and B cell precursors (1.42 ± 0.3; p = .011), but not abnormal PCs (1.09 ± 0.2; p = .352). There was no evidence that the recovery of cells was different between "Pre-lysis" versus "Dextran Sedimentation." All three flow cytometric assays achieved a minimum sensitivity of 10-5 and approached that of 10-6 for detecting rare events. CONCLUSION: Both "Pooled-tube" and "Dextran Sedimentation" staining procedures were comparable to the "Pre-lysis" method and are suitable high sensitivity flow cytometric approaches that can be used to process bone marrow samples for MM MRD testing.
Assuntos
Citometria de Fluxo , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/patologia , Idoso , Idoso de 80 Anos ou mais , Biópsia por Agulha , Medula Óssea/imunologia , Medula Óssea/patologia , Separação Celular/métodos , Separação Celular/normas , Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Humanos , Imunofenotipagem/métodos , Imunofenotipagem/normas , Pessoa de Meia-Idade , Monitorização Fisiológica/métodos , Monitorização Fisiológica/normas , Mieloma Múltiplo/terapia , Metástase Neoplásica , Neoplasia Residual , Plasmócitos/imunologia , Plasmócitos/patologia , Recidiva , Sensibilidade e EspecificidadeRESUMO
Mass cytometry, or CyTOF, is a useful technology for high-parameter single-cell phenotyping, especially from suspension cells such as blood or PBMC. It is particularly appealing to monitor the systemic immune changes that could accompany cancer immunotherapy. Here we present a reference panel for identification of all major immune cell populations, with flexibility for addition of trial-specific markers. We also describe best-practice measures for minimizing and tracking batch variability. These include: sample barcoding, use of spiked-in reference cells, and lyophilization of the antibody cocktail. Our protocol assumes the use of cryopreserved PBMC, both for convenience of batching samples and for maximum comparability across patients and time points. Finally, we show an option for automated analysis using the Astrolabe platform (Astrolabe Diagnostics, Inc.).
Assuntos
Anticorpos/imunologia , Coleta de Amostras Sanguíneas/métodos , Separação Celular/normas , Neoplasias/imunologia , Coleta de Amostras Sanguíneas/instrumentação , Citometria de Fluxo , Liofilização , Guias como Assunto , Humanos , Leucócitos Mononucleares , Espectrometria de Massas , Proteômica , Análise de Célula ÚnicaRESUMO
BACKGROUND AIMS: Mesenchymal stromal cells (MSC) have gained prominence in the field of regenerative medicine due to their excellent safety profile in human patients and recently demonstrated efficacy in late-stage clinical studies. A prerequisite to achieving successful MSC-based therapies is the development of large-scale manufacturing processes that preserve the biological potency of the founder cell population. Because no standardized manufacturing process exists for MSCs, understanding differences in these processes among U.S. academic facilities would allow for better comparison of results obtained in the clinical setting. METHODS: We collected information through a questionnaire sent to U.S. academic centers that produce MSCs under Good Manufacturing Practice conditions. RESULTS: The survey provided information on the number and geographic location of academic facilities in the United States and major trends in their manufacturing practices. For example, most facilities employed MSCs enriched from bone marrow by plastic adherence and expanded in media supplemented with pooled human platelet lysate. Sterility testing and product identification via cell surface phenotype analysis were commonly reported practices, whereas initial and working cell plating densities, culture duration, product formulation and the intended use of the MSC product were highly variable among facilities. The survey also revealed that although most facilities assessed product potency, the methods used were limited in scope compared with the broad array of intended clinical applications of the product. CONCLUSIONS: Survey responses reported herein offer insight into the current best practices used to manufacture MSC-based products in the United States and how these practices may affect product quality and potency. The responses also provide a foundation to establish standardized manufacturing platforms.
Assuntos
Técnicas de Cultura de Células/normas , Células-Tronco Mesenquimais/citologia , Centros Médicos Acadêmicos/normas , Células da Medula Óssea/citologia , Contagem de Células , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Proliferação de Células , Separação Celular/métodos , Separação Celular/normas , Humanos , Controle de Qualidade , Inquéritos e Questionários , Estados UnidosAssuntos
Separação Celular , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Diferenciação Celular , Separação Celular/métodos , Separação Celular/normas , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/fisiologia , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/fisiologia , Controle de Qualidade , Engenharia Tecidual/métodos , Engenharia Tecidual/normasRESUMO
To date, different methods of isolation of amniotic stem cells have been developed. Our previous studies have demonstrated that there are significant differences in viability and efficiency of the isolation and culture process depending on the enzyme and medium used. The aim of this study was to present efficient protocol, which can be used within good manufacturing practise conditions. Amniotic membranes were obtained from ten woman 31-39 years old who signed informed constent. GMP regulations are applicable. The described protocol aims to obtain a clinically significant cell yield (>1*108). The cells may be maintained in the growth phase even for 2 months. The mesenchymal cells constitute about 75-95% of the cells in primary culture. Supervisory authorities require repetitive and reproducible laboratory protocol for stem cells culture. Presented protocol allow achieving clinically significant cell yield (>1*108) in 4-5 weeks. Cells can be transplanted as suspension or cell sheet.
Assuntos
Âmnio/citologia , Separação Celular/métodos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Cultura Primária de Células/métodos , Adulto , Separação Celular/normas , Células Cultivadas , Técnicas de Laboratório Clínico/métodos , Feminino , Humanos , Transplante de Células-Tronco Mesenquimais/métodos , Transplante de Células-Tronco Mesenquimais/normas , Gravidez , Cultura Primária de Células/normas , Reprodutibilidade dos Testes , Coleta de Tecidos e Órgãos/métodos , Coleta de Tecidos e Órgãos/normasRESUMO
Liquid biopsy represents an alternative to conventional biopsies for the evaluation of tumors mainly due to its easy sampling. One of the main applications is the enumeration of Circulating Tumor Cells (CTCs) to evaluate tumor progression or response to treatment. The analysis of the functional characteristics of CTCs could give us much more information about their role in order to establish a more personalized treatment for the patients. The major issue that has to be solved is the isolation of the CTC population. Multiple protocols have been developed, however none of them has demonstrated to be the definitive one. In fact, a combination of these techniques has often been performed in order to obtain a purer and viable population of CTCs. In this review we have summarized for the first time the different combinatorial approaches used in the last years to optimize the isolation of CTCs and their limitations.
Assuntos
Separação Celular , Células Neoplásicas Circulantes/patologia , Animais , Biópsia/métodos , Separação Celular/métodos , Separação Celular/normas , HumanosRESUMO
BACKGROUND: Flow cytometric (FC) analysis of intestinal tissue biopsies requires prompt cell isolation and processing to prevent cell death and generate valid data. We examined the effect of storage conditions prior to cell isolation and FC on viable cell yield and the proportions of immune cell phenotypes from intestinal biopsies. METHODS: Biopsies (Nâ¯=â¯224) from inflamed or non-inflamed ileal and/or colonic tissue from three patients with Crohn's disease were processed and analyzed immediately in duplicate, or stored under different conditions. Cells were isolated and stained for specific markers, followed by FC. RESULTS: Decreased mean live CD45+ cell counts were observed after storage of biopsies at -80⯰C dimethyl sulfoxide (DMSO)/citrate buffer compared with immediate processing (1794.3 vs. 19,672.7; pâ¯=â¯0.006]). A non-significant decrease in CD45+ live cell count occurred after storage at -20⯰C in DMSO/citrate buffer and cell yield was adequate for subsequent analysis. CD3+ cell proportions were significantly lower after storage at 4⯰C in complete medium for 48â¯h compared with immediate analysis. Mean CD14+ cell proportions were significantly higher after storage of biopsies at -80⯰C in DMSO/citrate buffer compared with immediate analysis (2.61% vs. 1.31%, pâ¯=â¯0.007). CD4+, CD8+ and CD4+/CD8+ cell proportions were unaffected by storage condition. CONCLUSION: Storage of intestinal tissue biopsies at -20⯰C in DMSO/citrate buffer for up to 48â¯h resulted in sufficient viable cell yield for FC analysis without affecting subsequent marker-positive cell proportions. These findings support the potential shipping and storage of intestinal biopsies for centralized FC analysis in multicenter clinical trials.
Assuntos
Separação Celular/normas , Citometria de Fluxo/normas , Mucosa Intestinal/citologia , Intestinos/patologia , Manejo de Espécimes/normas , Adulto , Biomarcadores , Biópsia , Contagem de Células/normas , Doença de Crohn/diagnóstico , Dimetil Sulfóxido , Feminino , Congelamento , Humanos , Estudos Prospectivos , Adulto JovemRESUMO
Access to treatment with CAR-T Cells at European hospitals in general and at French hospitals in particular remains limited, when compared with the situation that prevails in the USA or in certain Asian countries. Multiple reasons explain why European investigators lag behind their US or Chinese colleagues in this clinical research area. Some of these reasons are related to the European and French regulatory landscapes that hamper the design and rapid implementation of organizational solutions needed for safe and efficient administration of CAR-T Cells. We here identify some of these pressing issues and propose some possible paths to move forward.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Atenção à Saúde/legislação & jurisprudência , Atenção à Saúde/organização & administração , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos/uso terapêutico , Linfócitos T/transplante , Coleta de Tecidos e Órgãos , Separação Celular/métodos , Separação Celular/normas , Terapia Baseada em Transplante de Células e Tecidos/métodos , Terapia Baseada em Transplante de Células e Tecidos/normas , Ensaios Clínicos como Assunto/legislação & jurisprudência , Ensaios Clínicos como Assunto/métodos , Ensaios Clínicos como Assunto/organização & administração , Ensaios Clínicos como Assunto/normas , Comércio , Atenção à Saúde/normas , França , Humanos , Imunoterapia Adotiva/legislação & jurisprudência , Imunoterapia Adotiva/métodos , Imunoterapia Adotiva/normas , Legislação Médica , Coleta de Tecidos e Órgãos/legislação & jurisprudência , Coleta de Tecidos e Órgãos/métodos , Coleta de Tecidos e Órgãos/normasRESUMO
BACKGROUND: We have recently shown that memory B cells from murine CMV immune donor animals adoptively transferred into immunodeficient mice were highly effective in protecting from a viral infection indicating a therapeutic potential of virus specific memory B cells. These preclinical data provided evidence that a cell-based strategy supporting the humoral immune response might be effective in a clinical setting of immunodeficiency after allogeneic hematopoietic stem cell transplantation. As adoptive transfer of B cells has not been used before in a clinical setting it was necessary to establish a technology for the generation of good manufacturing practice (GMP)-grade B cell products. METHODS: Starting from the leukapheresis product of healthy blood donors, B cells were purified by two different separation strategies using GMP-grade microbeads and the CliniMACS system. A one-step protocol was used for positive enrichment of B lymphocytes with anti-CD19 microbeads. In a two-step enrichment protocol, first T lymphocytes were depleted by anti-CD3 microbeads and the remaining fraction was positively selected by anti-CD19 microbeads. RESULTS: The purity and recovery after enrichment of B lymphocytes from the leukapheresis material in both separations strategies was not statistically different. However, contamination of the B-cell product with T cells was significantly lower after the two-step protocol (0.16%, range 0.01-0.43% after two-step separation and 0.55%, range 0.28-0.85% after one-step separation, p < 0.05). Therefore, a combined CD3 depletion and CD19 enrichment was used for the production of GMP-conform B-cell products from the leukapheresis material of 17 healthy stem cell donors. The absolute B-cell numbers obtained in the final product was 4.70 ± 3.64 × 108 with a purity of 95.98 ± 3.31% B lymphocytes and a recovery of 18.9 ± 10.6%. Importantly, the contamination with CD3+ T cells was extremely low in the final B- cell products (0.10 ± 0.20%). Purified B cells exhibited normal antibody production after in vitro stimulation and showed excellent viability after cryopreservation. CONCLUSIONS: A GMP-grade B-cell product can be obtained with high purity and very low T-cell contamination using the two-step enrichment protocol based on CliniMACS® technology.
Assuntos
Transferência Adotiva , Linfócitos B/metabolismo , Separação Celular/métodos , Separação Celular/normas , Transplante de Células-Tronco Hematopoéticas , Controle de Qualidade , Antígenos CD/metabolismo , Humanos , Imunoglobulina G/metabolismo , Imunofenotipagem , Transplante HomólogoRESUMO
The normal adult human female mammary gland is a bilayered structure consisting of an outer basal layer and two readily distinguished subsets of cells within the inner luminal layer. We now present a validated methodology for undertaking large-scale multi-parameter mass cytometric analyses of these cell types at single-cell resolution. In addition, we show how combining this approach with in vitro clonogenic assays of the proliferative and signaling responses of normal human mammary cells to epidermal growth factor (EGF) allows additional subsets with different EGF responses to be discerned. This included the identification of a subset of cells within the phenotypically defined luminal progenitor fraction that displays an elevated content of active caspase-3, including some that generate clones in vitro in response to EGF, with immunohistochemical evidence of their presence in situ in fixed preparations of normal human breast tissue.
Assuntos
Caspase 3/metabolismo , Separação Celular/métodos , Glândulas Mamárias Humanas/citologia , Análise de Célula Única/métodos , Separação Celular/normas , Fator de Crescimento Epidérmico/farmacologia , Feminino , Células HeLa , Humanos , Células MCF-7 , Glândulas Mamárias Humanas/metabolismo , Análise de Célula Única/normas , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
BACKGROUND AIMS: Autologous macrophage therapy represents a potentially significant therapeutic advance for the treatment of severe progressive liver cirrhosis. Administration of macrophages has been shown to reduce inflammation and drive fibrotic scar breakdown and tissue repair in relevant models. This therapeutic approach is being assessed for safety and feasibility in a first-in-human trial (MAcrophages Therapy for liver CirrHosis [MATCH] trial). METHODS: We outline the development and validation phases of GMP production. This includes use of the CliniMACS Prodigy cell sorting system to isolate CD14+ cells; optimizing macrophage culture conditions, assessing cellular identity, product purity, functional capability and determining the stability of the final cell product. RESULTS: The GMP-compliant macrophage products have a high level of purity and viability, and have a consistent phenotypic profile, expressing high levels of mature macrophage markers 25F9 and CD206 and low levels of CCR2. The macrophages demonstrate effective phagocytic capacity, are constitutively oriented to an anti-inflammatory profile and remain responsive to cytokine and TLR stimulation. The process validation shows that the cell product in excipient is remarkably robust, consistently passing the viability and phenotypic release criteria up to 48 hours after harvest. CONCLUSIONS: This is the first report of validation of a large-scale, fully Good Manufacturing Practice-compliant, autologous macrophage cell therapy product for the potential treatment of cirrhosis. Phenotypic and functional assays confirm that these cells remain functionally viable for up to 48 h, allowing significant flexibility in administration to patients.
Assuntos
Técnicas de Cultura de Células/métodos , Cirrose Hepática/terapia , Macrófagos/citologia , Fagocitose/fisiologia , Biomarcadores/metabolismo , Técnicas de Cultura de Células/normas , Separação Celular/métodos , Separação Celular/normas , Transplante de Células/métodos , Citocinas/farmacologia , Feminino , Humanos , Lectinas Tipo C/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Monócitos/citologia , Receptores CCR2/metabolismo , Receptores de Superfície Celular/metabolismoRESUMO
According to the Committee for Advanced Therapies, amniotic stem cells were classified as an advanced therapy medicinal product. This work aims to standardize the isolation of amniotic stem cells and the selection of the optimal time of transplantation and cell application methods in burn patients according to the guidelines of the Good Manufacturing Practice. The placenta used in the study was sourced during a Cesarean section. The remnants of the amnion preparation were placed in a sterile container and transferred to a class B environment, where the primary cultures began. The highest average number of cells was obtained by tissue homogenization and culture growth on the AmnioGrow medium. The isolation of the pure monoculture should be performed using the antibodies against CD105. On the basis of an analysis of population doubling, the aging of a population, the cells' viability, and the severity of injury, the cells should be used between passages 3 and 6. Significant differences were found in the number and viability of cells that were transferred as a full sheet, depending on the transfer method. To sum up, amniotic cells are a promising source in the treatment of burns and can be used as a hospital exemption.
Assuntos
Âmnio/citologia , Queimaduras/terapia , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células/normas , Proliferação de Células , Separação Celular/métodos , Separação Celular/normas , Sobrevivência Celular , Células Cultivadas , Feminino , Humanos , Guias de Prática Clínica como Assunto/normas , Gravidez , Fatores de Tempo , CicatrizaçãoRESUMO
BACKGROUND AIMS: Primary hematopoietic stem and progenitor cells (HSPCs) are key components of cell-based therapies for blood disorders and are thus the authentic substrate for related research. We propose that ubiquitous small-volume diagnostic samples represent a readily available and as yet untapped resource of primary patient-derived cells for cell- and gene-therapy studies. METHODS: In the present study we compare isolation and storage methods for HSPCs from normal and thalassemic small-volume blood samples, considering genotype, density-gradient versus lysis-based cell isolation and cryostorage media with different serum contents. Downstream analyses include viability, recovery, differentiation in semi-solid media and performance in liquid cultures and viral transductions. RESULTS: We demonstrate that HSPCs isolated either by ammonium-chloride potassium (ACK)-based lysis or by gradient isolation are suitable for functional analyses in clonogenic assays, high-level HSPC expansion and efficient lentiviral transduction. For cryostorage of cells, gradient isolation is superior to ACK lysis, and cryostorage in freezing media containing 50% fetal bovine serum demonstrated good results across all tested criteria. For assays on freshly isolated cells, ACK lysis performed similar to, and for thalassemic samples better than, gradient isolation, at a fraction of the cost and hands-on time. All isolation and storage methods show considerable variation within sample groups, but this is particularly acute for density gradient isolation of thalassemic samples. DISCUSSION: This study demonstrates the suitability of small-volume blood samples for storage and preclinical studies, opening up the research field of HSPC and gene therapy to any blood diagnostic laboratory with corresponding bioethics approval for experimental use of surplus material.
Assuntos
Coleta de Amostras Sanguíneas/métodos , Coleta de Amostras Sanguíneas/normas , Separação Celular/métodos , Separação Celular/normas , Terapia Baseada em Transplante de Células e Tecidos/métodos , Leucócitos/patologia , Talassemia/sangue , Preservação de Sangue/métodos , Preservação de Sangue/normas , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Criopreservação , Estudos de Viabilidade , Congelamento , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Contagem de Leucócitos , Leucócitos/fisiologia , Testes Sorológicos , Talassemia/patologiaRESUMO
INTRODUCTION: Autologous peripheral blood hematopoietic progenitor cell collection (A-HPCC) in pediatric patients is considered relatively safe although technically challenging. Very little is known regarding the incidence, risk factors and impact of procedure-related adverse events (AE) on pediatric A-HPCC outcomes. METHODS: Prospective 4.5-year review of AE associated with pediatric A-HPCC. AE were graded by severity and type. Potential demographic and procedural risk factors, and the impact on product quality, were compared by t-test, chi-square, and linear regression. RESULTS: Sixty-two children underwent 110 A-HPCC, including 36 (58%) under 20 kg. Fifty-five AE were documented in 25.4% A-HPCCs and 39% of children (citrate 25%, access 19%, technical 11%, cardiovascular 0%, allergic 1.8%). No AE were noted in children < 10 kg anticoagulated with heparin. Access and technical AE accounted for 73% of severe AE, with line-related problems underlying most technical AE (87.5%, P = 0.006). AE were more likely in older (P = 0.012), heavier patients (P = 0.02), who frequently required more than one A-HPCC (P = 0.012). In contrast, young children were more likely to experience citrate AE with gastrointestinal symptoms (median age, 6 years; P = 0.076). AE had no impact on CD34 collection rates; however, mean CD34 yields (4.2 vs. 20.4 million/kg; P = 0.0035) were decreased in patients with technical AE due to lower peripheral CD34 counts and a high number of aborted procedures (37%). CONCLUSION: Venous access and flow-related issues are a major factor associated with moderate and severe AE, effecting â¼10% of patients. AE are more frequent with increasing patient age, weight, and number of procedures. J. Clin. Apheresis 32:35-48, 2017. © 2016 Wiley Periodicals, Inc.
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Separação Celular/normas , Transplante de Células-Tronco de Sangue Periférico/efeitos adversos , Células-Tronco de Sangue Periférico/citologia , Adolescente , Fatores Etários , Antígenos CD34/análise , Antígenos CD34/sangue , Peso Corporal , Criança , Pré-Escolar , Humanos , Transplante de Células-Tronco de Sangue Periférico/métodos , Transplante AutólogoRESUMO
The aim of the study was to extend the potential use of human stem cells isolated from amniotic fluid in medical applications by confirming their high homogeneity and quality. Amniotic fluid samples were collected during amniocentesis from 165 women during pregnancy. The proliferation rate, clonogenicity, karyotype, aging process, pluripotent cell markers, expression of surface markers, and the potential to differentiate into adipose, bone and cartilage cells of hAFSCs were analyzed. Obtained results revealed that mesenchymal stem cells could be derived successfully from amniotic fluid, which exhibit properties comparable with MSCs of other origins. It is the first study, in which such a large group of patients was involved. Comprehensive statistical and biological analysis were conducted some of which clearly being innovative in relation to human amniotic fluid-derived stem cells. J. Cell. Biochem. 118: 116-126, 2017. © 2016 Wiley Periodicals, Inc.
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Líquido Amniótico , Separação Celular/métodos , Células-Tronco Pluripotentes , Adolescente , Adulto , Líquido Amniótico/citologia , Líquido Amniótico/metabolismo , Antígenos de Diferenciação/biossíntese , Proliferação de Células/fisiologia , Separação Celular/normas , Senescência Celular/fisiologia , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , GravidezRESUMO
BACKGROUND AIMS: Thymic-derived regulatory T cells (tTreg) are critical regulators of the immune system. Adoptive tTreg transfer is a curative therapy for murine models of autoimmunity, graft rejection, and graft-versus-host disease (GVHD). We previously completed a "first-in-human" clinical trial using in vitro expanded umbilical cord blood (UCB)-derived tTreg to prevent GVHD in patients undergoing UCB hematopoietic stem cell transplantation (HSCT). tTreg were safe and demonstrated clinical efficacy, but low yield prevented further dose escalation. METHODS: To optimize yield, we investigated the use of KT64/86 artificial antigen presenting cells (aAPCs) to expand tTreg and incorporated a single re-stimulation after day 12 in expansion culture. RESULTS: aAPCs increased UCB tTreg expansion greater than eightfold over CD3/28 stimulation. Re-stimulation with aAPCs increased UCB tTreg expansion an additional 20- to 30-fold. Re-stimulated human UCB tTreg ameliorated GVHD disease in a xenogeneic model. Following current Good Manufacturing Practice (cGMP) validation, a trial was conducted with tTreg. tTreg doses up to >30-fold higher compared with that obtained with anti-CD3/28 mAb coated-bead expansion and Foxp3 expression was stable during in vitro expansion and following transfer to patients. Increased expansion did not result in a senescent phenotype and GVHD was significantly reduced. DISCUSSION: Expansion culture with cGMP aAPCs and re-stimulation reproducibly generates sufficient numbers of UCB tTreg that exceeds the numbers of T effector cells in an UCB graft. The methodology supports future tTreg banking and is adaptable to tTreg expansion from HSC sources. Furthermore, because human leukocyte antigen matching is not required, allogeneic UCB tTreg may be a useful strategy for prevention of organ rejection and autoimmune disease.