Top-down tissue engineering aims to produce functional tissues using biomaterials as scaffolds, thus providing cues for cell proliferation and differentiation. Conversely, the bottom-up approach aims ...to precondition cells to form modular tissues units (building-blocks) represented by spheroids. In spheroid culture, adult stem cells are responsible for their extracellular matrix synthesis, re-creating structures at the tissue level. Spheroids from adult stem cells can be considered as organoids, since stem cells recapitulate differentiation pathways and also represent a promising approach for identifying new molecular targets (biomarkers) for diagnosis and therapy. Currently, spheroids can be used for scaffold-free (developmental engineering) or scaffold-based approaches. The scaffold promotes better spatial organization of individual spheroids and provides a defined geometry for their 3D assembly in larger and complex tissues. Furthermore, spheroids exhibit potent angiogenic and vasculogenic capacity and serve as efficient vascularization units in porous scaffolds for bone tissue engineering. An automated combinatorial approach that integrates spheroids into scaffolds is starting to be investigated for macro-scale tissue biofabrication.
Background
The SARS‐CoV‐2 pandemic has spurred an unparalleled scientific endeavor to elucidate the virus’ structure, infection mechanisms, and pathogenesis. Two‐dimensional culture systems have been ...instrumental in shedding light on numerous aspects of COVID‐19. However, these in vitro systems lack the physiological complexity to comprehend the infection process and explore treatment options. Three‐dimensional (3D) models have been proposed to fill the gap between 2D cultures and in vivo studies. Specifically, spheroids, composed of lung cell types, have been suggested for studying SARS‐CoV‐2 infection and serving as a drug screening platform.
Methods
3D lung spheroids were prepared by coculturing human alveolar or bronchial epithelial cells with human lung stromal cells. The morphology, size, and ultrastructure of spheroids before and after SARS‐CoV‐2 infection were analyzed using optical and electron microscopy. Immunohistochemistry was used to detect spike protein and, thus, the virus presence in the spheroids. Multiplex analysis elucidated the cytokine release after virus infection.
Results
The spheroids were stable and kept their size and morphology after SARS‐CoV‐2 infection despite the presence of multivesicular bodies, endoplasmic reticulum rearrangement, tubular compartment‐enclosed vesicles, and the accumulation of viral particles. The spheroid responded to the infection releasing IL‐6 and IL‐8 cytokines.
Conclusion
This study demonstrates that coculture spheroids of epithelial and stromal cells can serve as a cost‐effective infection model for the SARS‐CoV‐2 virus. We suggest using this 3D spheroid as a drug screening platform to explore new treatments related to the cytokines released during virus infection, especially for long COVID treatment.
Scientists have been working on potential treatments for SARS‐CoV‐2 (A) using 3D lung spheroids (B) formed by alveolar epithelial, bronchial, and stromal cells (C). Ultrastructural changes occur within the spheroid after SARS‐CoV‐2 infection, resulting in cytokines secretion (D). Spheroids can be used for drug screening (E) and drug design (F) specifically to tackle cytokines release in long‐COVID cases (G and H).
The pathological condition of obesity is accompanied by a dysfunctional adipose tissue. We postulate that subcutaneous, preperitoneal and visceral obese abdominal white adipose tissue depots could ...have stromal vascular fractions (SVF) with distinct composition and adipose stem cells (ASC) that would differentially account for the pathogenesis of obesity.
In order to evaluate the distribution of SVF subpopulations, samples of subcutaneous, preperitoneal and visceral adipose tissues from morbidly obese women (n = 12, BMI: 46.2±5.1 kg/m2) were collected during bariatric surgery, enzymatically digested and analyzed by flow cytometry (n = 12). ASC from all depots were evaluated for morphology, surface expression, ability to accumulate lipid after induction and cytokine secretion (n = 3).
A high content of preadipocytes was found in the SVF of subcutaneous depot (p = 0.0178). ASC from the three depots had similar fibroblastoid morphology with a homogeneous expression of CD34, CD146, CD105, CD73 and CD90. ASC from the visceral depot secreted the highest levels of IL-6, MCP-1 and G-CSF (p = 0.0278). Interestingly, preperitoneal ASC under lipid accumulation stimulus showed the lowest levels of all the secreted cytokines, except for adiponectin that was enhanced (p = 0.0278).
ASC from preperitoneal adipose tissue revealed the less pro-inflammatory properties, although it is an internal adipose depot. Conversely, ASC from visceral adipose tissue are the most pro-inflammatory. Therefore, ASC from subcutaneous, visceral and preperitoneal adipose depots could differentially contribute to the chronic inflammatory scenario of obesity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background:
Adipose tissue engineering may provide 3D models for the understanding of diseases such as obesity and type II diabetes. Recently, distinct adipose stem/stromal cell (ASC) subpopulations ...were identified from subcutaneous adipose tissue (SAT): superficial (sSAT), deep (dSAT), and the superficial retinacula cutis (sRC). This study aimed to test these subpopulations ASCs in 3D spheroid culture induced for adipogenesis under a pro-inflammatory stimulus with lipopolysaccharide (LPS).
Methods:
The samples of abdominal human subcutaneous adipose tissue were obtained during plastic aesthetic surgery (Protocol 145/09).
Results:
ASC spheroids showed high response to adipogenic induction in sSAT. All ASC spheroids increased their capacity to lipolysis under LPS. However, spheroids from dSAT were higher than from sSAT (
p
= 0.0045) and sRC (
p
= 0.0005). Newly formed spheroids and spheroids under LPS stimulus from sSAT showed the highest levels of fatty acid-binding protein 4 (FABP4) and CCAAT/enhancer-binding protein-α (C/EBPα) mRNA expression compared with dSAT and sRC (
p
< 0.0001). ASC spheroids from sRC showed the highest synthesis of angiogenic cytokines such as vascular endothelial growth factor (VEGF) compared with dSAT (
p
< 0.0228). Under LPS stimulus, ASC spheroids from sRC showed the highest synthesis of pro-inflammatory cytokines such as IL-6 compared with dSAT (
p
< 0.0092).
Conclusion:
Distinct physiological properties of SAT can be recapitulated in ASC spheroids. In summary, the ASC spheroid from dSAT showed the greatest lipolytic capacity, from sSAT the greatest adipogenic induction, and sRC showed greater secretory capacity when compared to the dSAT. Together, all these capacities form a true mimicry of SAT and hold the potential to contribute for a deeper understanding of cellular and molecular mechanisms in healthy and unhealthy adipose tissue scenarios or in response to pharmacological interventions.
Glioblastoma (GBM) is an infiltrative tumor that is difficult to eradicate. Treating GBM with mesenchymal stem cells (MSCs) that have been modified with the HSV-Tk suicide gene has brought ...significant advances mainly because MSCs are chemoattracted to GBM and kill tumor cells via a bystander effect. To use this strategy, abundantly present adipose-tissue-derived mesenchymal stem cells (AT-MSCs) were evaluated for the treatment of GBM in mice. AT-MSCs were prepared using a mechanical protocol to avoid contamination with animal protein and transduced with HSV-Tk via a lentiviral vector. The U-87 glioblastoma cells cultured with AT-MSC-HSV-Tk died in the presence of 25 or 50 μM ganciclovir (GCV). U-87 glioblastoma cells injected into the brains of nude mice generated tumors larger than 3.5 mm2 after 4 weeks, but the injection of AT-MSC-HSV-Tk cells one week after the U-87 injection, combined with GCV treatment, drastically reduced tumors to smaller than 0.5 mm2. Immunohistochemical analysis of the tumors showed the presence of AT-MSC-HSV-Tk cells only within the tumor and its vicinity, but not in other areas of the brain, showing chemoattraction between them. The abundance of AT-MSCs and the easier to obtain them mechanically are strong advantages when compared to using MSCs from other tissues.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
As an alternative to the classical tissue engineering approach, bottom‐up tissue engineering emerges using building blocks in bioassembly technologies. Spheroids can be used as building blocks to ...reach a highly complex ordered tissue by their fusion (bioassembly), representing the foundation of biofabrication. In this study, we analyzed the biomechanical properties and the fusion capacity of human adipose stem/stromal cell (ASC) we spheroids during an in vitro model of hypertrophic cartilage established by our research group. Hypertrophic induced‐ASC spheroids showed a statistically significant higher Young's modulus at weeks 2 (P < .001) and 3 (P < .0005) compared with non‐induced. After fusion, non‐induced and induced‐ASC spheroids increased the contact area and decreased their pairs' total length. At weeks 3 and 5, induced‐ASC spheroids did not fuse completely, and the cells migrate preferentially in the fusion contact region. Alizarin red O staining showed the highest intensity of staining in the fused induced‐ASC spheroids at week 5, together with intense staining for collagen type I and osteocalcin. Transmission electron microscopy and element content analysis (X‐ray Energy Dispersive Spectroscopy) revealed in the fused quartet at week 3 a crystal‐like structure. Hypertrophic induction interferes with the intrinsic capacity of spheroids to fuse. The measurements of contact between spheroids during the fusion process, together with the change in viscoelastic profile to the plastic, will impact the establishment of bioassembly protocols using hypertrophic induced‐ASC spheroids as building blocks in biofabrication.
The hypertrophic induction of adipose stem/stromal cells (ASC) spheroids positively impacts their biomechanical properties and alters their viscoelastic to plastic behavior, changing their building‐block capacity (fusion).
Fused spheroids showed an improvement in their osteogenesis.
Our findings have implications on bottom‐up tissue engineering opening a new perspective to establish bioassembly protocols in biofabrication using hypertrophic induced‐ASC spheroids as building blocks.
Background In human subcutaneous adipose tissue, the superficial fascia distinguishes superficial and deep microenvironments showing extensions called retinacula cutis. The superficial subcutaneous ...adipose tissue has been described as hyperplastic and the deep subcutaneous adipose tissue as inflammatory. However, few studies have described stromal-vascular fraction (SVF) content and adipose-derived stromal/stem cells (ASCs) behavior derived from superficial and deep subcutaneous adipose tissue. In this study, we analyzed a third conjunctive microenvironment: the retinacula cutis superficialis derived from superficial subcutaneous adipose tissue. Methods The samples of abdominal human subcutaneous adipose tissue were obtained during plastic aesthetic surgery in France (Declaration DC-2008-162) and Brazil (Protocol 145/09). Results The SVF content was characterized in situ by immunofluorescence and ex vivo by flow cytometry revealing a high content of pre-adipocytes rather in superficial subcutaneous adipose tissue microenvironment. Adipogenic assays revealed higher percentage of lipid accumulation area in ASCs from superficial subcutaneous adipose tissue compared with retinacula cutis superficialis (p < 0.0001) and deep subcutaneous adipose tissue (p < 0.0001). The high adipogenic potential of superficial subcutaneous adipose tissue was corroborated by an up-regulation of adipocyte fatty acid-binding protein (FABP4) compared with retinacula cutis superficialis (p < 0.0001) and deep subcutaneous adipose tissue (p < 0.0001) and of C/EBPalpha (CCAAT/enhancer-binding protein alpha) compared with retinacula cutis superficialis (p < 0.0001) and deep subcutaneous adipose tissue (p < 0.0001) microenvironments. Curiously, ASCs from retinacula cutis superficialis showed a higher level of adiponectin receptor gene compared with superficial subcutaneous adipose tissue (p = 0.0409), widely known as an anti-inflammatory hormone. Non-induced ASCs from retinacula cutis superficialis showed higher secretion of human vascular endothelial growth factor (VEGF), compared with superficial (p = 0.0485) and deep (p = 0.0112) subcutaneous adipose tissue and with adipogenic-induced ASCs from superficial (p = 0.0175) and deep (p = 0.0328) subcutaneous adipose tissue. Furthermore, ASCs from retinacula cutis superficialis showed higher secretion of Chemokine (C-C motif) ligand 5 (CCL5) compared with non-induced (p = 0.0029) and induced (p = 0.0089) superficial subcutaneous adipose tissue. Conclusions This study highlights the contribution to ASCs from retinacula cutis superficialis in their angiogenic property previously described for the whole superficial subcutaneous adipose tissue besides supporting its adipogenic potential for superficial subcutaneous adipose tissue. Keywords: Human subcutaneous adipose tissue, Superficial microenvironment, Deeper microenvironment, Retinacula cutis microenvironment, Stromal vascular fraction, Adipose stromal/stem cells
Human adipose stem/stromal cell (ASC) spheroids were used as a serum‐free in vitro model to recapitulate the molecular events and extracellular matrix organization that orchestrate a hypertrophic ...cartilage phenotype. Induced‐ASC spheroids (ø = 450 µm) showed high cell viability throughout the period of culture. The expression of collagen type X alpha 1 chain (COLXA1) and matrix metallopeptidase 13 (MMP‐13) was upregulated at week 2 in induced‐ASC spheroids compared with week 5 (P < .001) evaluated by quantitative real‐time PCR. In accordance, secreted levels of IL‐6 (P < .0001), IL‐8 (P < .0001), IL‐10 (P < .0001), bFGF (P < .001), VEGF (P < .0001), and RANTES (P < .0001) were the highest at week 2. Strong in situ staining for collagen type X and low staining for TSP‐1 was associated with the increase of hypertrophic genes expression at week 2 in induced‐ASC spheroids. Collagen type I, osteocalcin, biglycan, and tenascin C were detected at week 5 by in situ staining, in accordance with the highest expression of alkaline phosphatase (ALPL) gene and the presence of calcium deposits as evaluated by Alizarin Red O staining. Induced‐ASC spheroids showed a higher force required to compression at week 2 (P < .0001). The human ASC spheroids under serum‐free inducer medium and normoxic culture conditions were induced to a hypertrophic cartilage phenotype, opening a new perspective to recapitulate endochondral ossification in vivo.
The concept of "lockyballs" or interlockable mini-scaffolds fabricated by two-photon polymerization from biodegradable polymers for the encagement of tissue spheroids and their delivery into the ...desired location in the human body has been recently introduced. In order to improve control of delivery, positioning, and assembly of mini-scaffolds with tissue spheroids inside, they must be functionalized. This review describes the design, fabrication, and functionalization of mini-scaffolds as well as perspectives on their application in tissue engineering for precisely controlled cell and mini-tissue delivery and patterning. The development of functionalized mini-scaffolds advances the original concept of "lockyballs" and opens exciting new prospectives for mini-scaffolds' applications in tissue engineering and regenerative medicine and their eventual clinical translation.
To evaluate the short term safety and potential therapeutic effect of allogenic adipose tissue-derived stromal/stem cells (ASCs) + cholecalciferol in patients with recent-onset T1D.
Prospective, ...phase II, open trial, pilot study in which patients with recent onset T1D received ASCs (1 × 10
cells/kg) and cholecalciferol 2000 UI/day for 3 months (group 1) and were compared to controls with standard insulin therapy (group 2). Adverse events, C-peptide (CP), insulin dose, HbA1c, time in range (TIR), glucose variability (continuous glucose monitoring) and frequency of CD4
FoxP3+ T-cells (flow cytometry) were evaluated at baseline (T0) and after 3 months (T3).
13 patients were included (8: group 1; 5: group 2). Their mean age and disease duration were 26.7 ± 6.1 years and 2.9 ± 1.05 months. Adverse events were transient headache (
= 8), mild local reactions (
= 7), tachycardia (
= 4), abdominal cramps (
= 1), thrombophlebitis (
= 4), mild floaters (
= 2), central retinal vein occlusion (
= 1, complete resolution). At T3, group 1 had lower insulin requirement (0.22 ± 0.17 vs. 0.61±0.26IU/Kg;
= 0.01) and HbA1c (6.47 ± 0.86 vs. 7.48 ± 0.52%;
= 0.03) than group 2. In group 1, 2 patients became insulin free (for 4 and 8 weeks) and all were in honeymoon at T3 (vs. none in group 2;
= 0.01). CP variations did not differ between groups (-4.6 ± 29.1% vs. +2.3 ± 59.65%;
= 0.83).
Allogenic ASCs + cholecalciferol without immunosuppression was associated with stability of CP and unanticipated mild transient adverse events in patients with recent onset T1D.
NCT03920397.
PDF
