Technical challenges for complete implementation of automated growth-based methods for microbiological examination of advanced therapy medicinal products. What's wrong with Candida albicans?

BACKGROUND: Automated growth-based methods for sterility testing of cell-therapy products should be qualified to demonstrate that they are equivalent to, or better than, the conventional reference method. The aim of the present study was to assess the ability of the BACTEC FX40 system to detect low microbial contamination and to confirm the suitability of the method in the presence of seven different human mesenchymal cell-based products, according to Ph. Eur. 2.6.27. Additionally, a study to select the best vial to detect fungus contamination was performed. METHODS: Microorganisms representing Gram-negative, Gram-positive, aerobic, anaerobic, spore-forming, slow-growing bacteria, yeast and mold were prepared in either Dulbecco's PBS or seven biological matrices containing approximately 5, 10, and 15 colony-forming units (CFU) per sample. These preparations were inoculated to the specific media required for each test method: (i) BACTEC aerobic and anaerobic vials; (ii) aerobic and anaerobic media for direct inoculation; and (iii) Trypcase soy 3P or Brucella blood agar plates. Colonies from cultures were identified using MALDI-TOF mass spectrometry. RESULTS: The BACTEC FX40 system, in both Dulbecco's PBS and the biological matrices with a 5-CFU inoculum, detected most of the microorganisms significantly faster than the conventional method, despite the presence of a matrix containing gentamicin and several matrices containing 10% DMSO. Conversely, it showed an extremely delayed detection of Candida albicans compared with the conventional method. The addition of a Mycosis IC/F (MYC) vial decreased radically the time to detection (TTD) of C. albicans (28.2 ± 1.8 h) compared with the conventional method (36 h). CONCLUSIONS: When a MYC vial was added to the standard aerobic and anaerobic vials to test each sample, BACTEC FX40 was shown to be a superior alternative sterility method for cell-therapy products contaminated with low inocula, with a faster TTD for microbial growth compared with the conventional method (5 versus 14 days). The studies were carried out in different cell-based matrices with sensitivities and specificities of 100% for all the tested strains at 15-, 10- and 5-CFU inoculum, with the exception of Kocuria rhizophila at 5 CFU (90.48% sensitivity and 100% specificity).

Evaluation of the effectiveness of a new cryopreservation system based on a two-compartment vial for the cryopreservation of cell therapy products.

BACKGROUND AIMS: Successful cell cryopreservation and banking remain a major challenge for the manufacture of cell therapy products, particularly in relation to providing a hermetic, sterile cryovial that ensures optimal viability and stability post-thaw while minimizing exposure to toxic cryoprotective agents, typically dimethyl sulfoxide (Me2SO). METHODS: In the present study, the authors evaluated the effectiveness and functionality of Limbo technology (Cellulis S.L., Santoña, Spain). This system provides a hermetic vial with two compartments (one for adding cells with the cryoprotective agent solution and the other for the diluent solution) and an automated defrosting device. Limbo technology (Cellulis S.L.) allows reduction of the final amount of Me2SO, sidestepping washing and dilution steps and favoring standardization. The study was performed in several Good Manufacturing Practice laboratories manufacturing diverse cell therapy products (human mesenchymal stromal cells, hematopoietic progenitor cells, leukapheresis products, fibroblasts and induced pluripotent stem cells). Laboratories compared Limbo technology (Cellulis S.L.) with their standard cryopreservation procedure, analyzing cell recovery, viability, phenotype and functionality. RESULTS: Limbo technology (Cellulis S.L.) maintained the viability and functionality of most of the cell products and preserved sterility while reducing the final concentration of Me2SO. CONCLUSIONS: Results showed that use of Limbo technology (Cellulis S.L.) offers an overall safe alternative for cell banking and direct infusion of cryopreserved cell products into patients.

Endometrial and decidual stromal precursors show a different decidualization capacity.

Endometrial stromal cells (EnSCs) and decidual stromal cells (DSCs) originate from fibroblastic precursors located around the vessels of the human nonpregnant endometrium and the pregnant endometrium (decidua), respectively. Under the effect of ovarian or pregnancy hormones, these precursors differentiate (decidualize), changing their morphology and secreting factors that appear to be essential for the normal development of pregnancy. However, the different physiological context - that is, non-pregnancy vs pregnancy - of those precursors (preEnSCs, preDSCs) might affect their phenotype and functions. In the present study, we established preEnSC and preDSC lines and compared the antigen phenotype and responses to decidualization factors in these two types of stromal cell line. Analyses with flow cytometry showed that preEnSCs and preDSCs exhibited a similar antigen phenotype compatible with that of bone marrow mesenchymal stem/stromal cells. The response to decidualization in cultures with progesterone and cAMP was evaluated by analyzing changes in cell morphology by microscopy, prolactin and IL-15 secretion by enzyme immunoassay and the induction of apoptosis by flow cytometry. In all four analyses, preDSCs showed a significantly higher response than preEnSCs. The expression of progesterone receptor (PR), protein kinase A (PKA) and FOXO1 was studied with Western blotting. Both types of cells showed similar levels of PR and PKA, but the increase in PKA RI subunit expression in response to decidualization was again significantly greater in preDSCs. We conclude that preEnSCs and preDSCs are equivalent cells but differ in their ability to decidualize. Functional differences between them probably derive from factors in their different milieus.

Human predecidual stromal cells are mesenchymal stromal/stem cells and have a therapeutic effect in an immune-based mouse model of recurrent spontaneous abortion.

BACKGROUND: Human decidual stromal cells (DSCs) are involved in the maintenance and development of pregnancy, in which they play a key role in the induction of immunological maternal-fetal tolerance. Precursors of DSCs (preDSCs) are located around the vessels, and based on their antigen phenotype, previous studies suggested a relationship between preDSCs and mesenchymal stromal/stem cells (MSCs). This work aimed to further elucidate the MSC characteristics of preDSCs. METHODS: We established 15 human preDSC lines and 3 preDSC clones. Physiological differentiation (decidualization) of these cell lines and clones was carried out by in vitro culture with progesterone (P4) and cAMP. Decidualization was confirmed by the change in cellular morphology and prolactin (PRL) secretion, which was determined by enzyme immunoassay of the culture supernatants. We also studied MSC characteristics: (1) In mesenchymal differentiation, under appropriate culture conditions, these preDSC lines and clones differentiated into adipocytes, osteoblasts, and chondrocytes, and differentiation was confirmed by cytochemical assays and RT-PCR. (2) The expression of stem cell markers was determined by RT-PCR. (3) Cloning efficiency was evaluated by limited dilution. (4) Immunoregulatory activity in vivo was estimated in DBA/2-mated CBA/J female mice, a murine model of immune-based recurrent abortion. (5) Survival of preDSC in immunocompetent mice was analyzed by RT-PCR and flow cytometry. RESULTS: Under the effect of P4 and cAMP, the preDSC lines and clones decidualized in vitro: the cells became rounder and secreted PRL, a marker of physiological decidualization. PreDSC lines and clones also exhibited MSC characteristics. They differentiated into adipocytes, osteoblasts, and chondrocytes, and preDSC lines expressed stem cell markers OCT-4, NANOG, and ABCG2; exhibited a cloning efficiency of 4 to 15%; significantly reduced the embryo resorption rate (P < 0.001) in the mouse model of abortion; and survived for prolonged periods in immunocompetent mice. The fact that 3 preDSC clones underwent both decidualization and mesenchymal differentiation shows that the same type of cell exhibited both DSC and MSC characteristics. CONCLUSIONS: Together, our results confirm that preDSCs are decidual MSCs and suggest that these cells are involved in the mechanisms of maternal-fetal immune tolerance.

Characterization of mesenchymal stem/stromal cells with lymphoid tissue organizer cell potential in tonsils from children.

Lymphoid tissue organizer (LTo) cells, identified in mouse and human embryos, are thought to be precursors of stromal cells in secondary lymphoid organs. Whether LTo cells are present in human adults, however remains unknown. We obtained 15 stromal cell lines from tonsils from children who underwent tonsillectomy, and studied the antigen phenotype of these tonsil stromal cell (TSC) lines by flow cytometry and RT-PCR. Cell lines met the minimal criteria proposed by the International Society for Cellular Therapy to define human mesenchymal stem/stromal cells (MSCs): plastic-adherent capacity; expression of CD73, CD90 and CD105, lack of CD45, CD19 and HLA-DR; and capacity to differentiate into adipocytes, osteoblasts and chondrocytes. Furthermore, our TSC lines exhibited an antigen phenotype and functional characteristics very similar to those seen in murine embryo LTo cells: they expressed chemokines CCL19, CCL21 and CXCL13, cytokines TRANCE and IL-7, and adhesion molecules ICAM-1, mucosal addressin cell adhesion molecule (MadCAM)-1 and VCAM-1. The expression of LTo cell-associated markers and functions were upregulated by lymphotoxin (LT)α1ß2 and TNF, two cytokines involved in the development and maturation of secondary lymphoid tissues. Our results show that TSCs are tonsil MSCs that differentiate into LTo-like cells in response to the effects of these cytokines.

Human predecidual stromal cells have distinctive characteristics of pericytes: Cell contractility, chemotactic activity, and expression of pericyte markers and angiogenic factors.

INTRODUCTION: Human decidual stromal cells (DSCs) play a key role in maternal-fetal interactions. Precursors of DSCs (preDSCs) localize around vessels in both the endometrium and decidua. Previous studies suggested a relationship between preDSCs and pericytes because these cells share a perivascular location, alpha smooth muscle actin (α-SM actin) expression and the ability to contract under the effects of cytokines. METHODS: To further study this relationship, we established 15 human preDSC lines and 3 preDSC clones. The preDSC lines and clones were tested by flow cytometry with a panel of 29 monoclonal antibodies, 14 of which are pericyte markers. The expression of angiogenic factors was determined by RT-PCR, chemotactic activity was studied with the migration assay, and cell contractility was evaluated with the collagen cell contraction assay. Confocal microscopy was used to study decidual sections. RESULTS: Under the effect of progesterone and cAMP, these lines decidualized in vitro: the cells became rounder and secreted prolactin, a marker of physiological DSC differentiation (decidualization). The antigen phenotype of these preDSC lines and clones was fully compatible with that reported for pericytes. PreDSC lines displayed pericyte characteristics: they expressed angiogenic factors and showed chemotactic and cytokine-induced contractile activity. Confocal microscopic examination of decidual sections revealed the expression of antigens detected in preDSC lines: α-SM actin colocalized with CD146, CD140b, MFG-E8, nestin, and STRO-1 (all of which are pericyte markers) in cells located around the vessels, a distinctive location of preDSCs and pericytes. DISCUSSION: Taken together, our results show that preDSCs are pericyte-like cells.

Impaired B cells survival upon production of inflammatory cytokines by HIV-1 exposed follicular dendritic cells.

BACKGROUND: Follicular dendritic cells (FDCs) are important components in the organization of germinal centers in lymphoid tissue where, following antigen presentation, B cells differentiate into memory B cells. The possibility of establishing primary cell lines from FDCs isolated from lymphoid tissue paved the way for characterization of FDC biological properties. We exposed primary FDC cell lines to HIV-1 strains in vitro and studied changes in the chemo-attractive properties of FDCs and release of inflammatory cytokines. RESULTS: FDC lines expressed several known and putative HIV-1 receptors; viral genome was amplified in HIV-1 exposed FDCs which released low levels of p24 HIV-1 protein in culture supernatants, but were not definitely proven to be productively infected. Exposure of FDCs to HIV-1 strains did not change the expression of markers used to characterize these cells. HIV-1 exposed FDCs, however, changed the expression of chemo-attractants involved in cell recruitment at inflammatory sites and increased the production of several inflammatory cytokines. The inflammatory milieu created upon HIV-1 exposure of FDCs led to impaired B cell survival in vitro and reduced Ig production. CONCLUSIONS: FDC lines exposed to different HIV-1 strains, although not able to support productive HIV-1 replication, show an increased production of inflammatory cytokines. Our in vitro model of interactions between HIV-1 exposed FDC lines and B cells suggest that exposure of FDCs to HIV-1 in vivo can contribute to inflammation within germinal centers and that this pathological event may impair B cell survival and contribute to impaired B cell responses during HIV-1 infection.

Contractile activity of human follicular dendritic cells.

Follicular dendritic cells (FDCs) present antigens to B cells in the lymphoid follicle and inhibit B-cell apoptosis. In previous work, we obtained human FDC lines that allowed us to study the antigen phenotype and functions of these cells, finding that they expressed α-smooth muscle (SM) actin (a protein involved in cell contraction) and were able to contract collagen gel matrixes in gel contraction assays. Actin polymerization associated with cell contractility is essential for many cellular functions. We report here that interleukin (IL)-2 and interferon (IFN)-γ increased FDC contractility, and IL-10 reduced contractility, whereas IL-4 had no effect. Tumor necrosis factor (TNF) and lymphotoxin (LT)-α1ß2, cytokines involved in FDC differentiation, also increased FDC contractility. In different cell systems, cell contraction is related with the incorporation of α-SM actin into stress fibers. By confocal microscopy, we showed that cytochalasin D, an inhibitor of actin polymerization, inhibited α-SM actin incorporation and relaxed FDCs. Likewise, IL-10 significantly decreased the proportion of FDCs with α-SM actin-positive stress fibers, whereas cytokines that increased FDC contractility also increased this proportion. However, none of the cytokines tested significantly affected α-SM actin expression as determined by flow cytometry. IL-10, in addition to decreasing FDC contractility, increased the inhibitory activity of FDC in spontaneous B-cell apoptosis (P<0.05), but the other cytokines did not affect this activity. We conclude that cytokines related with FDC physiology regulate the contractility of these cells, and IL-10 also regulates the effect of FDC on B-cell apoptosis.

Liaison between natural killer cells and dendritic cells in human gestation.

A successful pregnancy relies on immunological adaptations that allow the fetus to grow and develop in the uterus, despite being recognized by maternal immune cells. Among several immunocompetent cell types present within the human maternal/fetal interface, DC-SIGN(+) dendritic cells (DCs) and CD56(+) natural killer (NK) cells are of major importance for early pregnancy maintenance, not only generating maternal immunological tolerance but also regulating stromal cell differentiation. Previous reports show the presence of NK-DC cell conjugates in first trimester human decidua, suggesting that these cells may play a role in the modulation of the local immune response within the uterus. While effective immunity is necessary to protect the mother from harmful pathogens, some form of tolerance must be activated to avoid an immune response against fetal antigens. This review article discusses current evidence concerning the functions of DC and NK cells in pregnancy and their liaison in human decidua.

Profiling Lgals9 splice variant expression at the fetal-maternal interface: implications in normal and pathological human pregnancy.

Disruption of fetal-maternal tolerance mechanisms can contribute to pregnancy complications, including spontaneous abortion. Galectin-9 (LGALS9), a tandem repeat lectin associated with immune modulation, is expressed in the endometrium during the mid and late secretory phases and in decidua during human early pregnancy. However, the role of LGALS9 during pregnancy remains poorly understood. We used real-time PCR and immunohistochemical staining to analyze the expression of Lgals9/LGALS9 during mouse gestation as well as in human tissues obtained from normal pregnancy and spontaneous abortions. In mice, three Lgals9 splice variants were detected, the expression of which was differentially regulated during gestation. Furthermore, decidual Lgals9 expression was deregulated in a mouse model of spontaneous abortion, whereas placental levels did not change. We further found that the LGALS9 D5 isoform suppresses interferon gamma production by decidual natural killer cells. In human patients, six Lgals9 splice variants were detected, and a decrease in Lgals9 D5/10 was associated with spontaneous abortion. Altogether, these results show a differential regulation of Lgals9 isoform expression during normal and pathological pregnancies and designate Lgals9 as a potential marker for adverse pregnancy outcomes.

Human decidual stromal cells express HLA-G: Effects of cytokines and decidualization.

BACKGROUND: Decidual stromal cells (DSC) are the main cellular component of the decidua, the maternal tissue in close contact with fetal trophoblast. Although of mesenchymal origin, DSC exert numerous immune functions that seem to be relevant for the immunological relationship between the mother and fetus. HLA-G, an antigen preferentially expressed by trophoblast, appears to participate in the immune tolerance by the mother of the semiallogeneic fetus. METHODS AND RESULTS: We show by flow cytometry, fluorescence microscopy, western blotting and RT-PCR that DSC isolated and maintained in culture express HLA-G weakly but consistently. We also detected this antigen by flow cytometry in fresh DSC. Interleukin (IL)-10, a cytokine associated with normal pregnancy, increased the expression of HLA-G by DSC (P < 0.00001), whereas IL-2, a cytokine involved in spontaneous abortion, showed no effect. Decidualization by progesterone and cAMP also up-regulated the expression of HLA-G by DSC (P < 0.001). Interferon gamma, a cytokine implicated in the vascular remodelling of the decidua necessary for embryo implantation, also increased the expression of HLA-G by DSC (P < 0.05). CONCLUSIONS: Our results suggest the existence of a network in which hormones together with cytokines regulate the expression of HLA-G by DSC, and that may be of relevance in the maintenance of maternal-fetal tolerance.

Follicular dendritic cells are related to bone marrow stromal cell progenitors and to myofibroblasts.

Follicular dendritic cells (FDC) are involved in the presentation of native Ags to B cells during the secondary immune response. Some authors consider FDC to be hemopoietic cells, whereas others believe them to be mesenchymal cells. The low proportion of FDC in the lymphoid follicle, together with technical difficulties in their isolation, make these cells difficult to study. We show that Fibroblast Medium can be used successfully to isolate and maintain FDC lines. In this culture medium, we obtained 18 FDC lines from human tonsils, which proliferated for as long as 18 wk and showed a stable Ag phenotype as detected by flow cytometry and RT-PCR. FDC lines were CD45-negative and expressed Ags associated to FDC (CD21, CD23, CD35, CD40, CD73, BAFF, ICAM-1, and VCAM-1) and Ags specific for FDC (DRC-1, CNA.42, and HJ2). These cell lines were also able to bind B cells and secrete CXCL13, functional activities characteristic of FDC. Nevertheless, the additional expression of STRO-1, together with CD10, CD13, CD29, CD34, CD63, CD73, CD90, ICAM-1, VCAM-1, HLA-DR, alkaline phosphatase, and alpha-smooth muscle actin (alpha-SM actin) indicated that FDC are closely related to bone marrow stromal cell progenitors. The expression of alpha-SM actin also relates FDC with myofibroblasts. Like myofibroblasts, FDC lines expressed stress fibers containing alpha-SM actin and were able to contract collagen gels under the effect of TGFbeta1 and platelet-derived growth factor. These findings suggest that FDC are a specialized form of myofibroblast and derive from bone marrow stromal cell progenitors.

Contractile activity of human decidual stromal cells. II. Effect of interleukin-10.

CONTEXT: Human decidual stromal cells (DSC) are myofibroblast-like cells that express alpha-smooth muscle (alpha-SM) actin, a protein associated with cell contractility. Several lines of experimental evidence in humans and mice show that antiinflammatory cytokines favor normal pregnancy, whereas Th1 and inflammatory cytokines play a role in abortion. We previously demonstrated that IL-2, a Th1 cytokine, increased the contractility of human DSC. OBJECTIVE: We studied the effect of the antiinflammatory cytokines IL-10 and IL-4 on the contractility of DSC from first-trimester pregnancy. SETTING AND PATIENTS: We studied 10 healthy women who underwent elective vaginal termination of first-trimester pregnancy at Clínica El Sur, Málaga, and Clínica Ginegranada, Granada. MAIN OUTCOME MEASURE(S): After isolation of DSC, cell contractility was measured with the collagen gel contraction assay. alpha-SM actin was detected with Western blotting and immunofluorescence. RESULTS: We found that IL-10, but not IL-4, increased the volume of the collagen gel matrixes in which the cytokine-treated DSC were cultured, showing that IL-10 decreased DSC contractility. By Western blotting we demonstrated that this effect was not related to an alteration in the synthesis of alpha-SM actin. Nevertheless, we observed by immunofluorescence microscopy that DSC treated with IL-10 exhibited stress fibers with a lower content of alpha-SM actin than untreated control DSC. CONCLUSIONS: IL-10 relaxes DSC by reducing the incorporation of alpha-SM actin into their stress fibers. This relaxing activity may be of relevance for the maintenance of pregnancy.