Engineered three-dimensional microenvironments as functional in vitro models of stromal tissues

Currently, the majority of current cultures is still carried out with long-established techniques like the exploitation of 2D supports, the use of tissue-derived immortalized cell lines, and the administration of un-physiological doses of soluble factors to induce a biological response. However, the lack of structural and physical cues often leads to biological artifacts, from the total loss of cellular function to the lack of correlation between the predicted and actual results when the experimental model shifts from in vitro to in vivo. Hence, in this work I test the hypothesis that recapitulating in vitro crucial chemo-physical components of the native cell environment can uniquely maintain the original function and the phenotype of cultured cells. Therefore, the critical aspects are (i) the choice of a suitable source of cells, and (ii) the engineering of the culture conditions. In first instance, it is proposed that freshly isolated adult cells, as opposed to cell lines, are needed to mimic physiological and pathological processes occurring in animal tissues and organs. Secondly, in vitro culture conditions need to be adapted to support cell viability, function, and growth. In particular, the proposed approach relies on the combination of the cells with a suitable biomaterial able to provide a 3D environment for cell adhesion and suitable to allow complex spatial interactions with neighboring cells. The concept of the third dimension as a critical parameter able to influence cell physiology is challenged in different contexts. The complexity of the proposed culture systems, due to the high number of variables among 2D and 3D experimental groups, is such that the precise dissection of the single contributions is not obvious. However, we propose that the combination of a physiological 3D architecture with a suitable biomaterial provide technological and biological advantages able to trigger further investigations. Notably, the material itself can be chosen so to mimic the native organ, e.g. the mineralized matrix of bone substituted in vitro by a ceramic material. Additionally, we suggest that the use of bioreactors as supportive technologies can exploit the full potential of 3D cell cultures. Despite implying an increase in the complexity of the procedures required to execute experiments based on 3D cell cultures, it is proposed that the relevance of the results surpasses the efforts required to implement new culture models. In the first chapter of my thesis, I focused on the validation of a platform for the expansion of bone-marrow derived stromal cells (MSC). As a result, the bioreactor-based platform was validated not only as a streamlined approach to expand MSC that maintain at a higher extent progenitor features, but also as a valuable tool to recreate in vitro an engineered stromal niche. In the second chapter of the thesis the focus was moved to exploit the unique features of 3D cultures on the recapitulation of the thymic stroma in vitro. This chapter describes the evolution of a culture system able to manufacture in vitro a thymic organoid constituted by TEC that can suits as a model to investigate thymus physiology. Finally, in the third chapter of the thesis, the concept of 3D stromal tissue engineering is applied to the hematopoietic niche, a specialized microenvironment devoted to regulate hematopoietic stem cells (HSC) quiescence and activity through a wide array of chemo-physical cues. Starting from previous reports in which freshly harvested bone marrow- or adipose tissue-derived cells can be cultured within porous scaffolds, allowing the formation of an organized 3D stromal tissue, we propose that cellularized constructs can be cultured in perfusion bioreactors to reconstruct the HSC niche through the controlled modulation of several parameters. Taken together, these results highlight that an increase in the complexity of the traditional culture systems is crucial to better recapitulate the functional microenvironment of stromal and stroma-dependent cells or stem cells. Growing and handling cells in a 3D structure combined with a compliant biomaterial and bioengineering tools can dramatically increase the relevance of scientific data, enable unpredecented modalities to control the artificial microenvironment, and decrease the need of costly, time consuming, and ethically debated in vivo experiments

In Memoriam: Paolo Bianco

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Engineering Small-Scale and Scaffold-Based Bone Organs via Endochondral Ossification Using Adult Progenitor Cells

Bone development, growth, and repair predominantly occur through the process of endochondral ossification, characterized by remodelling of cartilaginous templates. The same route efficiently supports engineering of bone marrow as a niche for hematopoietic stem cells (HSC). Here we describe a combined in vitro/in vivo system based on bone marrow-derived Mesenchymal Stem/Stromal Cells (MSC) that duplicates the hallmark cellular and molecular events of endochondral ossification during development. The model requires MSC culture with instructive molecules to generate hypertrophic cartilage tissues. The resulting constructs complete the endochondral route upon in vivo implantation, in the timeframe of up to 12 weeks. The described protocol is clearly distinct from the direct ossification approach typically used to drive MSC towards osteogenesis. Recapitulation of endochondral ossification allows modelling of stromal-HSC interactions in physiology and pathology and allows engineering processes underlying bone regeneration

An improved cartilage digestion method for research and clinical applications

Enzymatic isolation of chondrocytes from a cartilage biopsy is the first step to establish in vitro models of chondrogenesis or to generate cell-based grafts for cartilage repair. Such process is based on manually operated procedures and typically results in yields lower than 20% of the total available cells. In this study, we hypothesized that, as compared to conventionally used protocols, the enzymatic digestion of human articular cartilage in the presence of ascorbic acid 2-phosphate (AscA2P) or of sodium chloride (NaCl), in combination with the use of a perfusion bioreactor system, leads to a higher and more reproducible yield of cell populations with high proliferation and chondrogenic capacity. The addition of AscA2P within the enzymatic digestion medium did not significantly increase the cell yield, but resulted in a significant decrease of the intradonor variability in cell yield (-17.8% ± 10.7%, p = 0.0247) and in a significant increase of the proliferation rate of the isolated chondrocytes (+19.0% ± 1.4%, p < 0.05) with respect to the control group. The addition of NaCl during cartilage digestion did not modulate cell yield. When the cartilage digestion was further performed under direct perfusion flow, beneficial synergistic effects were achieved, with an overall increase of 34.7% ± 6.8% (p < 0.001) in the cell yield and an average decrease of 57.8% ± 11.2% (p < 0.01) in the coefficient of variation with respect to the control group. Importantly, by implementing this strategy it was possible to retrieve clonal subpopulations more efficiently capable of undergoing chondrogenesis, both in vitro and in vivo. Our findings bear relevance for the preparation of human chondrocytes for laboratory investigations, and in the perspective of efficient and streamlined manufacturing of cell/tissue grafts for articular cartilage repair

Reference values of urinary chromium in Italy

Objective: The paper describes the results of a polycentric study for the assessment of reference values of urinary chromium (U-Cr) in the Italian population. Method: A total of 890 subjects (58.3% males and 41.7% females) were selected on the basis of standardized criteria in eight different areas of Italy. Urinary chromium was determined on morning spot samples collected using standardized procedures. The U-Cr was determined independently by three laboratories using an Electrothermic atomization-Atomic Absorption Spectrometry (ETA-AAS) method with a detection limit of 0.05 mu g/l, adopting - for the statistical analysis - the median value of the results of the three laboratories. The between-laboratories within-subjects standard deviation was 0.049 mu g/l. Due to the high proportion (approx. 28%) of undetectable chromium levels, the geometric mean (GM) and geometric standard deviation (GSD) were estimated using a procedure of linear interpolation. The analysis of the effects of some variables (sex, age, center, residence, smoking and drinking habits) on the U-Cr values was also performed, by multiple regression analysis after logarithmic transformation, using GM and SD. Results: The reference value of U-Cr was of 0.08 mu g/l as an estimated GM, whereas the expected distribution ranged from not detectable (nd) (95% CI = nd-0.06) to 0.24 mu g/l (95th percentile; 95% CI = 0.20-0.31). Among the variables studied, only geographical area and sex significantly influenced the U-Cr levels. In subjects selected in the provinces of Bari and Venice values of U-Cr were significantly lower than those determined in subjects residing in other areas. Conclusions: From our investigation the reference values for U-Cr were lower than those obtained in previous investigations. In addition it confirms a further reduction in U-Cr levels following the previous decline reported in the 1970s and 1980s. In over 20 years U-Cr values in the general population dropped from values greater than 1 mu g/l to values between 0.5 and 0.2 mu g/l. The reasons of this progressive decline cannot be attributed in our opinion to a reduced intake of the metal, but mainly to the improvement in analytical instrumentation and methods. A further decrease may be ascribed to a more accurate definition of the reference groups and to a better control of pre-analytical factors. Considering that the reference values for U-Cr are much lower than those determined some decades ago, toxicological studies in order to verify the significance of biological limit values currently suggested for chromium seem to be necessary

Expansion of Human Mesenchymal Stromal Cells from Fresh Bone Marrow in a 3D Scaffold-Based System under Direct Perfusion

<div><p>Mesenchymal stromal/stem cell (MSC) expansion in conventional monolayer culture on plastic dishes (2D) leads to progressive loss of functionality and thus challenges fundamental studies on the physiology of skeletal progenitors, as well as translational applications for cellular therapy and molecular medicine. Here we demonstrate that 2D MSC expansion can be entirely bypassed by culturing freshly isolated bone marrow nucleated cells within 3D porous scaffolds in a perfusion-based bioreactor system. The 3D-perfusion system generated a stromal tissue that could be enzymatically treated to yield CD45- MSC. As compared to 2D-expanded MSC (control), those derived from 3D-perfusion culture after the same time (3 weeks) or a similar extent of proliferation (7–8 doublings) better maintained their progenitor properties, as assessed by a 4.3-fold higher clonogenicity and the superior differentiation capacity towards all typical mesenchymal lineages. Transcriptomic analysis of MSC from 5 donors validated the robustness of the process and indicated a reduced inter-donor variability and a significant upregulation of multipotency-related gene clusters following 3D-perfusion- as compared to 2D-expansion. Interestingly, the differences in functionality and transcriptomics between MSC expanded in 2D or under 3D-perfusion were only partially captured by cytofluorimetric analysis using conventional surface markers. The described system offers a multidisciplinary approach to study how factors of a 3D engineered niche regulate MSC function and, by streamlining conventional labor-intensive processes, is prone to automation and scalability within closed bioreactor systems.</p></div

Schematic overview of the experimental setup.

<p>Bone marrow aspirates were seeded into the 3D perfusion system and in conventional Petri dishes. After culture, cells from both systems were enzymatically retrieved and CD45- sorted cells using magnetic beads were analyzed as described.</p

Analysis of the expression of surface markers in 2D and 3D cultured MSC.

<p>Colored lines display the frequency of positive cells compared to isotype (gray lines). Most markers were similarly expressed in the two experimental groups. CD90, CD105, CD166, and ALP positive populations were more represented in monolayer culture, while CD146 and SSEA-1 were more represented in 3D-perfusion culture.</p

Phenotypical and growth characteristics for 2D and 3D perfused MSC.

<p>(a) Scanning electron microscopy imaging of cells within the scaffold display a complex network of branched fibroblastic-like adherent cells and the presence of rounded cells possibly of hematopoietic origin. (b) 2D cultured MSC display a typical flat fibroblastic morphology. (c) Flow cytometry of cultured cells shows a higher frequency of CD45+ cells in the perfusion system. (d) Proliferation rates indicate higher proliferation in 2D as compared to 3D perfusion cultured MSC. Statistically significant differences (P<0.05) are indicated with an asterisk (*; n = 5).</p