Comparing mechanical and enzymatic isolation procedures to isolate adipose-derived stromal vascular fraction: A systematic review.

The stromal vascular fraction of adipose tissue has gained popularity as regenerative therapy for tissue repair. Both enzymatic and mechanical intraoperative SVF isolation procedures exist. To date, the quest for the preferred isolation procedure persists, due to the absence of standardised yield measurements and a defined clinical threshold. This systematic review is an update of the systematic review published in 2018, where guidelines were proposed to improve and standardise SVF isolation procedures. An elaborate data search in MEDLINE (PubMed), EMBASE (Ovid) and the Cochrane Central Register of Controlled Trials was conducted from September 2016 to date. A total of 26 full-text articles met inclusion criteria, evaluating 33 isolation procedures (11 enzymatic and 22 mechanical). In general, enzymatic and mechanical SVF isolation procedures yield comparable outcomes concerning cell yield (2.3-18.0 × 105 resp. 0.03-26.7 × 105 cells/ml), and cell viability (70%-99% resp. 46%-97.5%), while mechanical procedures are more time consuming (8-20 min vs. 50-210 min) and cost-efficient. However, as most studies used poorly validated outcome measures on SVF characterisation, it still remains unclear which intraoperative SVF isolation method is preferred. Future studies are recommended to implement standardised guidelines to standardise methods and improve comparability between studies.

Phylogenomic perspectives on speciation and reproductive isolation in a North American biodiversity hotspot: an example using California sages (Salvia subgenus Audibertia: Lamiaceae).

BACKGROUND AND AIMS: The California Floristic Province (CA-FP) is the most species-rich region of North America north of Mexico. One of several proposed hypotheses explaining the exceptional diversity of the region is that the CA-FP harbours myriad recently diverged lineages with nascent reproductive barriers. Salvia subgenus Audibertia is a conspicuous element of the CA-FP, with multiple sympatric and compatible species. METHODS: Using 305 nuclear loci and both organellar genomes, we reconstruct species trees, examine genomic discordance, conduct divergence-time estimation, and analyse contemporaneous patterns of gene flow and mechanical reproductive isolation. KEY RESULTS: Despite strong genomic discordance, an underlying bifurcating tree is supported. Organellar genomes capture additional introgression events not detected in the nuclear genome. Most interfertility is found within clades, indicating that reproductive barriers arise with increasing genetic divergence. Species are generally not mechanically isolated, suggesting that it is unlikely to be the primary factor leading to reproductive isolation. CONCLUSIONS: Rapid, recent speciation with some interspecific gene flow in conjunction with the onset of a Mediterranean-like climate is the underlying cause of extant diversity in Salvia subgenus Audibertia. Speciation has largely not been facilitated by gene flow. Its signal in the nuclear genome seems to mostly be erased by backcrossing, but organellar genomes each capture different instances of historical gene flow, probably characteristic of many CA-FP lineages. Mechanical reproductive isolation appears to be only part of a mosaic of factors limiting gene flow.

A Simple and Effective Mechanical Method for Adipose-Derived Stromal Vascular Fraction Isolation.

Over the last decades, there has been ongoing and evolving research concerning regenerative medicine, specifically, stem cells. The most common source of adult mesenchymal stem cells (MSCs) remains the adipose tissue and the easiest way to obtain such tissue is lipoaspirate. The fatty tissue obtained can be processed either in an enzymatic way, which is time-consuming and expensive and carries several dangers for the viability of the stem cells included, or with mechanical means which are fast, inexpensive, yield enough viable cells, and can be readily used for autologous transplantation in one-stage procedures. Herein, we demonstrate our non-enzymatic method for obtaining adipose-derived stromal vascular fraction comprising MSCs. The stromal vascular fraction was isolated via centrifugation, and the characteristics and numbers of the cells isolated have been tested with flow cytometry assay, cell culture, and differentiation. Over 91% of viable MSCs were isolated using the mechanical method. The cells retained the ability to differentiate into osteocytes, adipocytes, and chondrocytes. The method presented is simple, requiring no special equipment, and yields a viable population of stem cells in large numbers. These cells can be readily used in several operations (orthopedic, dentistry, fistulas, etc.) making feasible "one-stage" procedures, thus proving their benefits for the patient and the health care system.

Method of Isolation and In Vitro Culture of Primordial Follicles in Bovine Animal Model.

The mammalian ovary is a substantial source of oocytes arranged into follicles at various stages of folliculogenesis, from the primordial to the ovulatory ones. Primordial follicles constitute the most abundant source of gametes inside the mammalian ovary at any given time.The isolation of a high number of primordial follicles, together with the development of protocols for in vitro follicle growth, would provide a powerful tool to fully exploit the female reproductive potential and boost the rescue and restoration of fertility in assisted reproduction technologies in human medicine, animal breeding, and preservation of threatened species. However, the most significant limitation is the lack of efficient methods for isolating a healthy and homogeneous population of viable primordial follicles suitable for in vitro culture. Here, we provide a fast and high-yield strategy for the mechanical isolation of primordial follicles from limited portions of the ovarian cortex in the bovine animal model.

Efficacy of stromal vascular fraction and enzyme-free mechanical isolation therapy in experimental full thickness burn wounds.

BACKGROUND: Autologous cell suspensions obtained by a stromal vascular fraction (SVF) and enzyme-free mechanical isolation (EMI) are an alternative in the treatment of burn wounds. In this study, we aimed to investigate the effect of autologous cell suspensions obtained by SVF and EMI on full-thickness skin burn wounds. METHODS: A total of 45 male Sprague-Dawley rats were divided into three groups, SVF group, EMI group, and SVF + EMI group. The groups were also classified as the first, second, and third week of the burn to reveal the effect of the treatment on the burn in the early, middle, and late stages. For treatment, 0.2 ml SVF or 0.2 ml EMI was injected subcutaneously into the burn lesions of the subjects. Histopathological examination was performed on the burn wounds taken at the end of the experiment, and Ki67, CD44, CD73, CD90, and CK17 expressions were evaluated. RESULTS: Histological examination revealed that there was no improvement in the control samples, but the skin was multicellular, vascularization was present. Histologic scores in all groups was significantly better than control, and SVF + EMI was the best group in terms of recovery (p < 0.05). Ki67, CK17, CD44, CD73, and CD90 expressions were significantly higher in the treatment groups compared to the control (p < 0.05). CONCLUSION: We found in our study that both applications significantly increased the healing of the burn wound. Moreover, SVF + EMI application provided more improvement than SVF or EMI alone.

Follicle isolation methods reveal plasticity of granulosa cell steroidogenic capacity during mouse in vitro follicle growth.

Follicles are the functional unit of the ovary and several methods have been developed to grow follicles ex vivo, which recapitulate key events of oogenesis and folliculogenesis. Enzymatic digestion protocols are often used to increase the yield of follicles from the ovary. However, the impact of these protocols on the outermost theca and granulosa cells, and thereby follicle function, is not well defined. To investigate the impact of enzymatic digestion on follicle function, we collected preantral follicles from CD1 mice either by enzymatic digestion (Enzy-FL) or mechanical isolation (Mech-FL) and compared follicle growth, steroidogenesis and cell differentiation within an encapsulated in vitro follicle growth system which maintains the 3D architecture of the oocyte and its surrounding somatic cells. Follicles were encapsulated in 0.5% alginate and cultured for 8 days. Compared with Enzy-FL, Mech-FL grew more rapidly and produced significantly higher levels of androstenedione, estradiol and progesterone. The expression of theca-interstitial cell marker genes, Cyp17a1, which encodes 17-hydroxylase/17, 20-lyase and catalyzes the hydroxylation of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, and the conversion of these products into dehydroepiandrosterone and androstenedione, and Star, which encodes a transport protein essential for cholesterol entry into mitochondria, were also higher in Mech-FL than in Enzy-FL. Mech-FL maintained an intact theca-interstitial layer on the outer edge of the follicle that phenocopied in vivo patterns as confirmed by alkaline phosphatase staining, whereas theca-interstitial cells were absent from Enzy-FL from the onset of culture. Therefore, preservation of the theca cell layer at the onset of culture better supports follicle growth and function. Interestingly, granulosa cells in the outermost layers of Enzy-FL expressed CYP17A1 by Day 4 of culture while maintaining inhibin α-subunit expression and a cuboidal nucleus. Thus, in the absence of theca-interstitial cells, granulosa cells have the potential to differentiate into androgen-producing cells. This work may have implications for human follicle culture, where enzymatic isolation is required owing to the density of the ovarian cortex.

Modified nanofat grafting: Stromal vascular fraction simple and efficient mechanical isolation technique and perspectives in clinical recellularization applications.

Background: Nanofat grafting (NG) is a simple and cost-effective method of lipoaspirates with inter-syringe passages, to produce stromal vascular fraction (SVF) and isolate adipose-derived stem cells (ASCs). This represents a tremendous interest in the future clinical needs of tissue engineering. In this study, we optimized the NG technique to increase the yield of ASC extractions. Methods: We analyzed three groups of SVF obtained by 20, 30, and 40 inter-syringe passages. The control group was an SVF obtained by enzymatic digestion with Celase. We studied their cell composition by flow cytometry, observed their architecture by confocal microscopy, and observed immunomodulatory properties of the ASCs from each of the SVFs by measuring inflammatory markers of macrophages obtained by an ASC monocyte co-culture. Results: We have established the first cell mapping of the stromal vascular fraction of adipose tissue. The results showed that SVF obtained by 20 inter-syringe passages contains more statistically significant total cells, more cells expressing the ASC phenotype, more endothelial cells, and produces more CFU-F than the SVF obtained by 30 and 40 passages and by enzymatic digestion. Confocal microscopy showed the presence of residual adipocytes in SVF obtained by inter-syringe passages but not by enzymatic digestion. The functional study indicates an orientation toward a more anti-inflammatory profile and homogenization of their immunomodulatory properties. Conclusion: This study places mechanically dissociated SVF in the center of approaches to easily extract ASCs and a wide variety and number of other progenitor cells, immediately available in a clinical setting to provide both the amount and quality of cells required for decellularized tissues.

A new method for primary culture of microglia in rats with spinal cord injury.

At present, the primary culture method of microglia is complicated, and the culture of spinal cord microglia is rare, so we will explore to establish a new and efficient primary culture method of microglia in rats with spinal cord injury (SCI). The SCI model of SD rats was established by modified A11en's method, and the model of SCI was performed on 1 d, 3 d, 7 d and 14 d respectively. Then the injured spinal cord was removed, mechanically separated and filtered. The morphology of microglia was observed the next day and its purity was identified by CD11b and Iba1 immunofluorescence labeling. According to the above results, the morphological changes of microglia after 3 d of SCI were observed at 1 d, 2 d and 4 d. The results showed that the purity of microglia was 98%. The number of microglia after 3 d of SCI was the most. After SCI, the migration ability of microglia was enhanced, the number of microglia in the injured area increased, and the number was the highest at 3 d, then gradually decreased. In addition, the microglia after SCI would gradually change from active state to resting state with the passage of time. Therefore, we can use a simple and efficient mechanical separation method to extract primary microglia, which provides the basis for the study of microglia.

In Vivo Evaluation of Mechanically Processed Stromal Vascular Fraction in a Chamber Vascularized by an Arteriovenous Shunt.

Mechanically processed stromal vascular fraction (mSVF) is a promising source for regenerative purposes. To study the in vivo fate of the mSVF, we herein used a vascularized tissue engineering chamber that insulates the target mSVF from the surrounding environment. In contrast to previous models, we propose an arteriovenous (AV) shunt between saphenous vessels in rats without a venous graft. Mechanical SVF was processed from the fat pads of male Sprague Dawley rats, mixed with a fibrin hydrogel and implanted into an inguinal tissue engineering chamber. An arteriovenous shunt was established between saphenous artery and vein. On the contralateral side, an mSVF-fibrin hydrogel mix without vascular axis served as a non-vascularized control. After two and six weeks, rats were sacrificed for further analysis. Mechanical SVF showed significant numbers of mesenchymal stromal cells. Vascularized mSVF explants gained weight over time. Perilipin and CD31 expression were significantly higher in the mSVF explants after six weeks while no difference in DAPI positive cells, collagen deposition and FABP4 expression was observed. Morphologically, no differentiated adipocytes but a dense cell-rich tissue with perilipin-positive cells was found after six weeks. The phosphorylation of ERK1/2 was significantly enhanced after six weeks while Akt activation remained unaltered. Finally, mSVF explants stably expressed and released VEGF, bFGF and TGFb. Vascularized mSVF is able to proliferate and express adipocyte-specific markers. The AV shunt model is a valuable refinement of currently existing AV loop models in the rat which contributes to the fundamental 3R principles of animal research.

Not Stromal Vascular Fraction (SVF) or Nanofat, but Total Stromal-Cells (TOST): A New Definition. Systemic Review of Mechanical Stromal-Cell Extraction Techniques.

The most important and greatest source in the body for regenerative cells is fat tissue. Obtaining regenerative cells from adipose tissue can be done in two ways: Enzymatic and mechanical. The regenerative cell cocktail obtained by the enzymatic method, including stem cells, is called Stromal vascular fracture (SVF). In the literature, there is no clear definition of regenerative cells obtained by mechanical method. We systematically searched the techniques and definitions for stromal cells obtained from adipose tissue by scanning different databases. To evaluate the mechanical stromal-cell isolation techniques and end products from adipose tissue. Systematic review of English and non-English articles using Embase, PubMed, Web of Science and Google scholar databases. Search terms included Nanofat, fragmented fat, mechanical stromal / stem cell, mechanical SVF, SVF gel. We screened all peer-reviewed articles related with mechanical stromal-cell isolation. Author performed a literature query with the aforementioned key words and databases. A total of 276 publications containing the keywords we searched were reached. In these publications, there are 46 different definitions used to obtain mechanical stromal cells. The term SVF is only suitable for enzymatic methods. A different definition is required for mechanical. The most used term nanofat is also not suitable because the product is not in both "fat" and in "nanoscale". We think that the term total stromal-cells would be the most appropriate definition since both extracellular matrix and all stromal cells are protected in mechanical methods.

Xenogen-free isolation and culture of human adipose mesenchymal stem cells.

BACKGROUND: Adipose-derived Stem Cells (ASCs) present great potential for reconstructive procedures. Currently, isolation by enzyme digestion and culturing using xenogenic substances remain the gold standard, impairing clinical use. METHODS: Abdominal lipo-aspirate and blood samples were obtained from healthy patients. A novel mechanical isolation method for ASCs was compared to (the standard) collagenase digestion. ASCs are examined by flowcytometry and multilineage differentiation assays. Cell cultures were performed without xenogenic or toxic substances, using autologous plasma extracted from peripheral blood. After eGFP-transfection, an in vivo differentiation assay was performed. RESULTS: Mechanical isolation is more successful in isolating CD34+/CD31-/CD45-/CD13+/CD73+/CD146- ASCs from lipo-aspirate than isolation via collagenase digestion (p <0 .05). ASCs display multilineage differentiation potential in vitro. Autologous plasma is a valid additive for ASCs culturing. eGFP-ASCs, retrieved after 3 months in vivo, differentiated in adipocytes and endothelial cells. CONCLUSION: A practical method for human ASC isolation and culturing from abdominal lipo-aspirate, without the addition of xenogenic substances, is described. The mechanical protocol is more successful than the current gold standard protocol of enzyme digestion. These results are important in the translation of laboratory-based cell cultures to clinical reconstructive and aesthetic applications.

Intraspecific divergence in floral-tube length promotes asymmetric pollen movement and reproductive isolation.

The causative link between phenotypic divergence and reproductive isolation is an important but poorly understood part of ecological speciation. We studied the effects of floral-tube length variation on pollen placement/receipt positions and reproductive isolation. In a population of Lapeirousia anceps (Iridaceae) with bimodal floral-tube lengths, we labelled pollen of short- and long-tubed flowers with different colour fluorescent nanoparticles (quantum dots). This enabled us to map pollen placement by long- and short-tubed flowers on the only floral visitor, a long-proboscid fly. Furthermore, it allowed us to quantify pollen movement within and between short- and long-tubed flowers. Short- and long-tubed flowers placed pollen on different parts of the pollinator, and long-tubed flowers placed more pollen per visit than short-tubed flowers. This resulted in assortative pollen receipt (most pollen received comes from the same phenotype) and strong but asymmetric reproductive isolation, where short-tubed plants are more reproductively isolated than long-tubed plants. These results suggest that floral-tube length divergence can promote mechanical isolation in plants through divergence in pollen placement sites on pollinators. Consequently, in concert with other reproductive isolation mechanisms, selection for differences in floral-tube length can play an important role in ecological speciation of plants.

Both morph- and species-dependent asymmetries affect reproductive barriers between heterostylous species.

The interaction between floral traits and reproductive isolation is crucial to explaining the extraordinary diversity of angiosperms. Heterostyly, a complex floral polymorphism that optimizes outcrossing, evolved repeatedly and has been shown to accelerate diversification in primroses, yet its potential influence on isolating mechanisms remains unexplored. Furthermore, the relative contribution of pre- versus postmating barriers to reproductive isolation is still debated. No experimental study has yet evaluated the possible effects of heterostyly on pre- and postmating reproductive mechanisms. We quantify multiple reproductive barriers between the heterostylous Primula elatior (oxlip) and P. vulgaris (primrose), which readily hybridize when co-occurring, and test whether traits of heterostyly contribute to reproductive barriers in unique ways. We find that premating isolation is key for both species, while postmating isolation is considerable only for P. vulgaris; ecogeographic isolation is crucial for both species, while phenological, seed developmental, and hybrid sterility barriers are also important in P. vulgaris, implicating sympatrically higher gene flow into P. elatior. We document for the first time that, in addition to the aforementioned species-dependent asymmetries, morph-dependent asymmetries affect reproductive barriers between heterostylous species. Indeed, the interspecific decrease of reciprocity between high sexual organs of complementary floral morphs limits interspecific pollen transfer from anthers of short-styled flowers to stigmas of long-styled flowers, while higher reciprocity between low sexual organs favors introgression over isolation from anthers of long-styled flowers to stigmas of short-styled flowers. Finally, intramorph incompatibility persists across species boundaries, but is weakened in long-styled flowers of P. elatior, opening a possible backdoor to gene flow through intramorph pollen transfer between species. Therefore, patterns of gene flow across species boundaries are likely affected by floral morph composition of adjacent populations. To summarize, our study highlights the general importance of premating isolation and newly illustrates that both morph- and species-dependent asymmetries shape boundaries between heterostylous species.

Nonadaptive radiation in damselflies.

Adaptive radiations have long served as living libraries to study the build-up of species richness; however, they do not provide good models for radiations that exhibit negligible adaptive disparity. Here, we review work on damselflies to argue that nonadaptive mechanisms were predominant in the radiation of this group and have driven species divergence through sexual selection arising from male-female mating interactions. Three damselfly genera (Calopteryx,Enallagma and Ischnura) are highlighted and the extent of (i) adaptive ecological divergence in niche use and (ii) nonadaptive differentiation in characters associated with reproduction (e.g. sexual morphology and behaviours) was evaluated. We demonstrate that species diversification in the genus Calopteryx is caused by nonadaptive divergence in coloration and behaviour affecting premating isolation, and structural differentiation in reproductive morphology affecting postmating isolation. Similarly, the vast majority of diversification events in the sister genera Enallagma and Ischnura are entirely driven by differentiation in genital structures used in species recognition. The finding that closely related species can show negligible ecological differences yet are completely reproductively isolated suggests that the evolution of reproductive isolation can be uncoupled from niche-based divergent natural selection, challenging traditional niche models of species coexistence.

Pollination in a patchily distributed lousewort is facilitated by presence of a co-flowering plant due to enhancement of quantity and quality of pollinator visits.

BACKGROUND AND AIMS: Plants surrounded by individuals of other co-flowering species may suffer a reproductive cost from interspecific pollen transfer (IPT). However, differences in floral architecture may reduce or eliminate IPT. METHODS: A study was made of Pedicularis densispica (lousewort) and its common co-flowering species, Astragalus pastorius, to compare reproductive and pollination success of lousewort plants from pure and mixed patches. Floral architecture and pollinator behaviour on flowers of the two plants were compared along with the composition of stigmatic pollen load of the louseworts. The extent of pollen limitation of plants from pure and mixed patches was also explored through supplemental pollination with self- and outcross pollen (PLs and PLx). KEY RESULTS: Mixed patches attracted many more nectar-searching individuals of Bombus richardsi. These bumble-bees moved frequently between flowers of the two species. However, they pollinated P. densispica with their dorsum and A. pastorius with their abdomen. This difference in handling almost completely eliminated IPT. Lousewort plants from mixed patches yielded more seeds, and seeds of higher mass and germinability, than those from pure patches. Moreover, louseworts from mixed patches had lower PLs and PLx compared with those from pure patches. CONCLUSIONS: Differences in floral architecture induced differences in pollinator behaviour that minimized IPT, such that co-flowering plants significantly enhanced quantity and quality of pollinator visits for the lousewort plants in patchy habitat. These findings add to our understanding of the mechanisms of pollination facilitation.