Indication-based protocols with different solutions for mechanical stromal-cell transfer

Background Regenerative medicine is the fastest developing branch of plastic surgery in recent times. Adipose tissue is one of the largest and most important sources in the body for stromal cells. Although mechanical isolation methods are both very popular and have many advantages, they still have no accepted protocols. Objective We developed new protocols called indication-based protocols (IPs) for standardization and new techniques called mechanical stromal-cell transfer (MEST) by using ultra-sharp blades and dilution of adipose tissue with different solutions (saline, Ringer and 5% Dextrose) Methods & material: In order to obtain the desired physical structure (liquid, gel, solid) and the desired volume, four different types of IPs have been defined. Adipose tissue was prediluted with different solutions using 10 or 20 cc injectors in IPs 1 and 2, while condensed adipose tissue was used directly in IPs 3 and 4. Results In MEST, stromal cells were obtained from 100 mL of condensed fat using different IPs with 92% mean viability and cell counts of 26.80–91.90 × 106. Stromal cells can be obtained in the desired form and number of cells by using four different IPs. Conclusion Isolation of stromal cells by cutting fat with sharp blades will prevent the death of fat tissue and stromal cells and will allow high viability and cell count with our new technique. Predilution with different solutions: Diluting the condensed adipose tissue with the desired solutions (saline, Ringer or 5% Dextrose) before the adinizing process will provide even more stromal cells. Lay Summary Obtaining regenerative stromal cells from adipose tissue can be done by two methods: Enzymatic and mechanical. Mechanical methods have many advantages. Although mechanical stromal cell extraction from adipose tissue is very popular and many techniques have been described, there are still no accepted protocols, definition for the end product, and no consensus on the status of the stromal cells. In this study, stromal cells were obtained mechanically by using ultra-sharp blade systems, without exposing adipose tissue to blunt trauma. Thus, a higher number of cells and higher viability could be obtained. An “Indication based” protocol has been defined for the first time in order to obtain the desired number and status (solid, semi-solid, liquid) end product. Diluting the condensed adipose tissue with the desired solutions (saline, Ringer or 5% Dextrose) before the adinizing process will provide even more stromal cells. This will provide an opportunity for clinicians to obtain and apply a stromal cell solution for different indications in different anatomical regions.

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New Mechanical Fat Separation Technique: Adjustable Regenerative Adipose-tissue Transfer (ARAT) and Mechanical Stromal Cell Transfer (MEST)

Aesthetic Surgery Journal Open Forum ◽

10.1093/asjof/ojaa035 ◽

2020 ◽

Vol 2 (4) ◽

Cited By ~ 1

Author(s):

H Eray Copcu ◽

Sule Oztan

Keyword(s):

Adipose Tissue ◽

Stromal Cells ◽

Stromal Cell ◽

Retention Rate ◽

High Rate ◽

Cell Transfer ◽

Fat Tissue ◽

Cell Counts ◽

Tissue Transfer ◽

Regenerative Cells

Abstract Background Adipose tissue is not only a very important source of filler but also the body’s greatest source of regenerative cells. Objectives In this study, adipose tissue was cut to the desired dimensions using ultra-sharp blade systems to avoid excessive blunt pressure and applied to various anatomical areas—a procedure known as adjustable regenerative adipose-tissue transfer (ARAT). Mechanical stromal cell transfer (MEST) of regenerative cells from fat tissue was also examined. Methods ARAT, MEST, or a combination of these was applied in the facial area of a total of 24 patients who were followed for at least 24 months. The integrity of the fat tissue cut with different diameter blades is shown histopathologically. The number and viability of the stromal cells obtained were evaluated and secretome analyses were performed. Patient and surgeon satisfaction were assessed with a visual analog scale. Results With the ARAT technique, the desired size fat grafts were obtained between 4000- and 200-micron diameters and applied at varying depths to different aesthetic units of the face, and a guide was developed. In MEST, stromal cells were obtained from 100 mL of condensed fat using different indication-based protocols with 93% mean viability and cell counts of 28.66 to 88.88 × 106. Conclusions There are 2 main complications in fat grafting: visibility in thin skin and a low retention rate. The ARAT technique can be used to prevent these 2 complications. MEST, on the other hand, obtains a high rate of fat and viable stromal cells without applying excessive blunt pressure. Level of Evidence: 4

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Cell Therapy in a Mouse Model of Immune-Mediated Bone Marrow Failure.

Blood ◽

10.1182/blood.v110.11.1689.1689 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1689-1689

Author(s):

Jichun Chen ◽

Neal S. Young

Keyword(s):

Bone Marrow ◽

T Cells ◽

Progenitor Cells ◽

Stromal Cells ◽

Stromal Cell ◽

Adherent Cells ◽

Hematopoietic Stem ◽

Cell Counts ◽

Immune Mediated ◽

Stem And Progenitor Cells

Abstract Immune-mediated bone marrow (BM) failure has been modeled in the mouse by infusion of lymph node cells from allogeneic C57BL/6 (B6) donors into major or minor histocompatibility antigen-mismatched recipients (Chen et al., Blood 2004; Bloom et al., Exp Hematol 2004, Chen et al., J Immunol 2007). Co-infusion of limited numbers of CD4+CD25+ regulatory T lymphocytes (Tregs) can alleviate clinical manifestations by suppressing the expansion of pathogenic T cells (Chen et al., J Immunol 2007). In the current study, we investigated the effectiveness of Tregs and suppressor cells contained in BM stroma in this fatal disease. Infusion of fewer than 3 × 103 Tregs to each recipient mouse had only a minor effect in preserving BM cells and did not prevent pancytopenia. Fifteen-50 × 103 thymic Tregs was moderately protective: blood WBC, RBC, platelet and BM cell counts at three weeks after cell infusion were 197%, 116%, 155% and 158% of those of control animals that did not receive Treg infusion; 5–10 × 103 B6 splenic Tregs produced the largest effect as WBC, RBC, platelet and BM cell counts were 275%, 143%, 276%, and 198% of controls. Overall, Treg therapy was helpful but its effectiveness was limited and variable among individual recipients as no antigen-specific Tregs can be identified for the treatment of BM failure. Learned about the immunosuppressive effects of mesenchymal stem cells (MSCs), we went on to test the effectiveness of stromal cells as another therapeutic modality for BM failure, since stromal cells contain MSCs. These cells were derived from B6 BM by culture in α-modified Eagle medium at 33°C with 5% CO2 for two weeks. After separating the non-adherent cells, we detached the adherent stromal cells and infused them into TBI + B6 LN-infused C.B10 mice. Injection of 106 stromal cells at the time of LN cell infusion effectively preserved WBCs (3.09 ± 0.51 vs 0.61 ± 0.18), RBCs (8.72 ± 0.14 vs 3.52 ± 0.46), platelets (924 ± 93 vs 147 ± 25) and BM cells (186.6 ± 8.7 vs 52.7 ± 7.8) when compared to LN-cell-infused mice without stromal cell addition. Delayed stromal cell injection at day 9 after LN cell infusion had only a mild effect on the preservation of RBCs (147%), platelets (276%) and BM cells (223%) and no effect on WBCs (64%), and infusion of non-adherent cells from the same stromal cell culture had no therapeutic effect. Stromal cell-infused mice had higher proportion of FoxP3+CD4+ cells in the peripheral blood (59.7 ± 10.7% vs 29.8 ± 5.4%) and more Lin−CD117+CD34− hematopoietic stem and progenitor cells in the BM (591 ± 95 vs 60 ± 43, thousand) in comparison to LN cell infused mice without stromal cell treatment. Mitigation of pathogenic T cells, including both CD4 and CD8 T lymphocytes, is the potential mechanism for the effectiveness of Treg and stromal cell therapies that helped to protect hematopoietic stem and progenitor cells in the BM of affected animals. Figure Figure

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In Vitro Characterization of Adipose Stem Cells Non-Enzymatically Extracted from the Thigh and Abdomen

International Journal of Molecular Sciences ◽

10.3390/ijms21093081 ◽

2020 ◽

Vol 21 (9) ◽

pp. 3081 ◽

Cited By ~ 1

Author(s):

Elena Dai Prè ◽

Alice Busato ◽

Silvia Mannucci ◽

Federica Vurro ◽

Francesco De Francesco ◽

...

Keyword(s):

Adipose Tissue ◽

Automatic Device ◽

Cell Number ◽

Fat Grafting ◽

The Body ◽

In Vitro Characterization ◽

Cellular Debris ◽

Mechanical Methods

Autologous fat grafting is a surgical technique in which adipose tissue is transferred from one area of the body to another, in order to reconstruct or regenerate damaged or injured tissues. Before reinjection, adipose tissue needs to be purified from blood and cellular debris to avoid inflammation and preserve the graft viability. To perform this purification, different enzymatic and mechanical methods can be used. In this study, we characterized in vitro the product of a closed automatic device based on mechanical disaggregation, named Rigenera®, focusing on two sites of adipose tissue harvesting. At first, we optimized the Rigenera® operating timing, demonstrating that 60 s of treatment allows a higher cellular yield, in terms of the cell number and growth rate. This result optimizes the mechanical disaggregation and it can increase the clinical efficiency of the final product. When comparing the extracted adipose samples from the thigh and abdomen, our results showed that the thigh provides a higher number of mesenchymal-like cells, with a faster replication rate and a higher ability to form colonies. We can conclude that by collecting adipose tissue from the thigh and treating it with the Rigenera® device for 60 s, it is possible to obtain the most efficient product.

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Adipose Stromal Cell Expansion and Exhaustion: Mechanisms and Consequences

Cells ◽

10.3390/cells9040863 ◽

2020 ◽

Vol 9 (4) ◽

pp. 863 ◽

Cited By ~ 3

Author(s):

Kristin Eckel-Mahan ◽

Aleix Ribas Latre ◽

Mikhail G. Kolonin

Keyword(s):

Adipose Tissue ◽

Stromal Cells ◽

Cell Expansion ◽

Stromal Cell ◽

Cell Types ◽

Mixed Population ◽

Adipose Stromal Cells ◽

Diet Induced Obesity ◽

Adipocyte Hypertrophy ◽

Adipose Stromal Cell

Adipose tissue (AT) is comprised of a diverse number of cell types, including adipocytes, stromal cells, endothelial cells, and infiltrating leukocytes. Adipose stromal cells (ASCs) are a mixed population containing adipose progenitor cells (APCs) as well as fibro-inflammatory precursors and cells supporting the vasculature. There is growing evidence that the ability of ASCs to renew and undergo adipogenesis into new, healthy adipocytes is a hallmark of healthy fat, preventing disease-inducing adipocyte hypertrophy and the spillover of lipids into other organs, such as the liver and muscles. However, there is building evidence indicating that the ability for ASCs to self-renew is not infinite. With rates of ASC proliferation and adipogenesis tightly controlled by diet and the circadian clock, the capacity to maintain healthy AT via the generation of new, healthy adipocytes appears to be tightly regulated. Here, we review the contributions of ASCs to the maintenance of distinct adipocyte pools as well as pathogenic fibroblasts in cancer and fibrosis. We also discuss aging and diet-induced obesity as factors that might lead to ASC senescence, and the consequences for metabolic health.

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Bone Marrow Stromal Cells Inhibit Apoptosis of Chronic Lymphocytic Leukemia Cells (CLL) by Expressing Erythroid Differentiation Regulator 1 (ERDR1)

Blood ◽

10.1182/blood.v118.21.1764.1764 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1764-1764

Author(s):

Hendrik W van Deventer ◽

Todd Hoffert ◽

Michelle L West ◽

Qing Ping Wu ◽

Jonathan S Serody

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Stromal Cell ◽

Mesenchymal Cells ◽

Lymphocytic Leukemia ◽

Wild Type ◽

Cell Counts ◽

Stromal Cell Line ◽

And Control

Abstract Abstract 1764 Background: Chemotherapy resistance in chronic lymphocytic leukemia (CLL) is in part mediated by anti-apoptotic signals produced by bone marrow stromal cells. Identifying these signals is the first step to overcoming this resistance. ERDR1 was initially described as an inducer of hemoglobinization. We now present evidence that it inhibits apoptosis of CLL cells. Methods/Results: Previously, we showed that wild type (WT) but not CCR5−/− mesenchymal cells increase pulmonary melanoma metastasis in CCR5−/− mice. This observation led us to compare gene expression in the lungs of these mice. Using an Affymetrix expression array, we found Erdr1 was differentially expressed in the wild type mice. To show that the increase in metastasis was mediated by Erdr1, we transferred WT pulmonary mesenchymal cells transfected with Erdr1 shRNA or non-targeted control shRNA. CCR5−/− mice receiving Erdr1 knockdown mesenchymal cells had a 27.3% to 37% decrease in metastasis compared to animals receiving control transfected cells (p<0.01). One explanation for the decrease in metastasis would be a failure of the Erdr1 knockdown cells to survive in the lung. Since the knockdown and control vectors express EGFP, we were able to compare the quantity of transfected cells surviving in the lung by applying an EGFP ELISA to lung homogenates. These experiments showed no difference in the number of surviving mesenchymal cells. These results suggested Erdr1 was acting as a pro-survival factor for the melanoma cells. Since ERDR1 expression is the highest in the bone marrow, we compared the survival of CLL cells co-cultured with control and Erdr1 knockdown cells. In these experiments, stable Erdr1 knockdown and control clones were selected after the transfection of the bone marrow stromal cell line M2-10B4. Peripheral blood samples were then collected from 10 untreated CLL patients and co-cultured with these stromal cell lines. After 72 and 96 hours, total cell counts and apoptosis were measured using Annexin V/PI. At both time points, the cell counts were higher when the CLL cells were co-cultured with control cell lines compared to Erdr1 knockdown lines (OR 1.88 ± 0.27, 2.52 ± 0.66 respectively). The increase in total cell number was associated with a decrease in the percentage of apoptotic cells (OR 0.69 ± 0.18, 0.58 ± 0.12 respectively). Since Erdr1 was differentially expressed in WT compared to CCR5−/− mice, we considered the regulation of this gene by chemokine agonists. In these experiments, 100 ng/ml of CCL4 was added to WT and CCR5−/− PMCs and mRNA was harvested at 12, 24, and 48 hours. Using real-time PCR, we found that Erdr1 expression was increased compared to baseline in the WT mesenchymal cells by 1.33 fold ± 0.06 (p < 0.05) after 24 hours. By 48 hours, expression had increased by 3.36 fold ± 0.14 (p < 0.001). As expected, CCL4 did not increase expression of Erdr1 in CCR5−/− mesenchymal cells. Since Erdr1 is associated with hemoglobinization, we also investigated the effect of hypoxia on Erdr1 expression. In these experiments, deferoxamine was added to the stromal cell line M2-10B4 at varying concentrations. Significant fold increases in Erdr1 expression were seen after 48 hours of 1.43 ± 0.10 (50nM), 1.96 ± 0.08, (100nM), and 2.44 ± 0.01 (200nM). Implications for the treatment of human disease: Other investigators have shown that stromal cells such as nurse-like cells and CLL cells can interact to induce CCL4 and promote CLL cell survival. Our work suggests a novel pathway by which this may take place. Disclosures: No relevant conflicts of interest to declare.

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Systemic Dermatitis Model Mice Exhibit Atrophy of Visceral Adipose Tissue and Increase Stromal Cells via Skin-Derived Inflammatory Cytokines

International Journal of Molecular Sciences ◽

10.3390/ijms21093367 ◽

2020 ◽

Vol 21 (9) ◽

pp. 3367

Author(s):

Kento Mizutani ◽

Eri Shirakami ◽

Masako Ichishi ◽

Yoshiaki Matsushima ◽

Ai Umaoka ◽

...

Keyword(s):

Adipose Tissue ◽

Inflammatory Cytokines ◽

Stromal Cells ◽

Skin Disease ◽

Skin Lesions ◽

The Body ◽

Whole Body ◽

Inflammatory Skin Disease ◽

Tnf Α ◽

Inflammatory Skin

Adipose tissue (AT) is the largest endocrine organ, producing bioactive products called adipocytokines, which regulate several metabolic pathways, especially in inflammatory conditions. On the other hand, there is evidence that chronic inflammatory skin disease is closely associated with vascular sclerotic changes, cardiomegaly, and severe systemic amyloidosis in multiple organs. In psoriasis, a common chronic intractable inflammatory skin disease, several studies have shown that adipokine levels are associated with disease severity. Chronic skin disease is also associated with metabolic syndrome, including abnormal tissue remodeling; however, the mechanism is still unclear. We addressed this problem using keratin 14-specific caspase-1 overexpressing transgenic (KCASP1Tg) mice with severe erosive dermatitis from 8 weeks of age, followed by re-epithelization. The whole body and gonadal white AT (GWAT) weights were decreased. Each adipocyte was large in number, small in size and irregularly shaped; abundant inflammatory cells, including activated CD4+ or CD8+ T cells and toll-like receptor 4/CD11b-positive activated monocytes, infiltrated into the GWAT. We assumed that inflammatory cytokine production in skin lesions was the key factor for this lymphocyte/monocyte activation and AT dysregulation. We tested our hypothesis that the AT in a mouse dermatitis model shows an impaired thermogenesis ability due to systemic inflammation. After exposure to 4 °C, the mRNA expression of the thermogenic gene uncoupling protein 1 in adipocytes was elevated; however, the body temperature of the KCASP1Tg mice decreased rapidly, revealing an impaired thermogenesis ability of the AT due to atrophy. Tumor necrosis factor (TNF)-α, IL-1β and interferon (INF)-γ levels were significantly increased in KCASP1Tg mouse ear skin lesions. To investigate the direct effects of these cytokines, BL/6 wild mice were administered intraperitoneal TNF-α, IL-1β and INF-γ injections, which resulted in small adipocytes with abundant stromal cell infiltration, suggesting those cytokines have a synergistic effect on adipocytes. The systemic dermatitis model mice showed atrophy of AT and increased stromal cells. These findings were reproducible by the intraperitoneal administration of inflammatory cytokines whose production was increased in inflamed skin lesions.

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Non-Epithelial Thymic Stromal Cells: Unsung Heroes in Thymus Organogenesis and T Cell Development

Frontiers in Immunology ◽

10.3389/fimmu.2020.620894 ◽

2021 ◽

Vol 11 ◽

Author(s):

Takeshi Nitta ◽

Hiroshi Takayanagi

Keyword(s):

T Cell ◽

Stromal Cells ◽

Stromal Cell ◽

T Cell Development ◽

Cell Types ◽

Cell Development ◽

The Body ◽

Thymic Epithelial Cells ◽

T Cell Repertoire ◽

Cell Repertoire

The stromal microenvironment in the thymus is essential for generating a functional T cell repertoire. Thymic epithelial cells (TECs) are numerically and phenotypically one of the most prominent stromal cell types in the thymus, and have been recognized as one of most unusual cell types in the body by virtue of their unique functions in the course of the positive and negative selection of developing T cells. In addition to TECs, there are other stromal cell types of mesenchymal origin, such as fibroblasts and endothelial cells. These mesenchymal stromal cells are not only components of the parenchymal and vascular architecture, but also have a pivotal role in controlling TEC development, although their functions have been less extensively explored than TECs. Here, we review both the historical studies on and recent advances in our understanding of the contribution of such non-TEC stromal cells to thymic organogenesis and T cell development. In particular, we highlight the recently discovered functional effect of thymic fibroblasts on T cell repertoire selection.

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A stromal cell niche sustains ILC2-mediated type-2 conditioning in adipose tissue

Journal of Experimental Medicine ◽

10.1084/jem.20190689 ◽

2019 ◽

Vol 216 (9) ◽

pp. 1999-2009 ◽

Cited By ~ 26

Author(s):

Batika M.J. Rana ◽

Eric Jou ◽

Jillian L. Barlow ◽

Noe Rodriguez-Rodriguez ◽

Jennifer A. Walker ◽

...

Keyword(s):

Adipose Tissue ◽

Immune Cells ◽

Stromal Cells ◽

Stromal Cell ◽

Cell Stress ◽

Lymphoid Cells ◽

Type 2 Cytokines ◽

Group 2 ◽

Cell Niche

Group-2 innate lymphoid cells (ILC2), type-2 cytokines, and eosinophils have all been implicated in sustaining adipose tissue homeostasis. However, the interplay between the stroma and adipose-resident immune cells is less well understood. We identify that white adipose tissue–resident multipotent stromal cells (WAT-MSCs) can act as a reservoir for IL-33, especially after cell stress, but also provide additional signals for sustaining ILC2. Indeed, we demonstrate that WAT-MSCs also support ICAM-1–mediated proliferation and activation of LFA-1–expressing ILC2s. Consequently, ILC2-derived IL-4 and IL-13 feed back to induce eotaxin secretion from WAT-MSCs, supporting eosinophil recruitment. Thus, MSCs provide a niche for multifaceted dialogue with ILC2 to sustain a type-2 immune environment in WAT.

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