Name: isolation, characterization, and therapeutic application of extracellular vesicles from cultured human mesenchymal stem cells
Uploaded: 2022-09-23T13:00:00
Description: Watch this Scientific Journal Video about Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells at JoVE.com

This work was supported by grants from the National Natural Science Foundation of China (32000974, 81930025, and 82170988) and the China Postdoctoral Science Foundation (2019M663986 and BX20190380). We are grateful for the assistance of the National Experimental Teaching Demonstration Center for Basic Medicine (AMFU) and the Analytical and Testing Central Laboratory of Military Medical Innovation Center of Air Force Medical University.

All animal procedures were approved by the Animal Care and Use Committee of the Fourth Military Medical University and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Eight-week-old C57Bl/6 mice (no preference for either females or males) were used. Cryopreserved human umbilical cord-derived MSCs (UCMSCs), used for the present study, were obtained from a commercial source (see Table of Materials). The use of human cells was approved by the ...

<ol>
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EVs are emerging to play an important role in diverse biological activities, including antigen presentation, genetic material transport, cell microenvironment modification, and others. Furthermore, their wide application brings new approaches and opportunities for diagnosing and treating diseases21. Implementation of therapeutic applications of EVs is based on successful isolation and characterization. However, due to the lack of standardized isolation and purification methods and the low extracti...

Stem cells are undifferentiated pluripotent cells with self-renewal capability and translational potential1. Mesenchymal stem cells (MSCs) are easily isolated, cultured, expanded, and purified in the laboratory, which remains characteristic of stem cells after multiple passages. In recent years, increasing evidence has supported the view that MSCs act in a paracrine mode in therapeutic use2,3. Especially the secretion of extracellular vesicles (EVs) plays a crucial role in mediating the biological functions of MSCs. As heterogeneous membranous nanoparticles released from most cell types, EVs consist of subcategories termed exosomes (Exos), microvesicles (MVs), and even larger apoptotic bodies4,5. Among them, Exos is the most widely studied EV with a size of 40-150 nm, which is of an endosomal origin and actively secreted in physiological conditions. MVs are formed by shedding directly from the surface of the cell plasma membrane with a diameter of 100-1,000 nm, which are characterized by high expression of phosphatidylserine and expression of surface markers of donor cells6. EVs contain RNA, proteins, and other bioactive molecules, which have similar functions to the parent cells and play a significant role in cell communication, immune response, and tissue damage repair7. MSC-EVs have been widely investigated as a powerful cell-free therapeutic tool in regenerative medicine8.
Isolation and purification of MSC-derived EVs is a common issue in the field of research and application. At present, differential and density gradient ultracentrifugation9, ultrafiltration process10, immunomagnetic separation11, molecular exclusion chromatograph12, and microfluidic chip13 are widely employed methods in the isolation and purification of EVs. With the advantages and disadvantages of each approach, the quantity, purity, and activity of collected EVs cannot be satisfied at the same time14,15. In the present study, the differential centrifugation protocol of isolation and characterization of EVs from cultured MSCs is shown in detail, which has supported efficient therapeutic use16,17,18,19,20. The adaptability of this method for a series of downstream approaches, such as fluorescent labeling, local transplantation, and systemic injection, has further been exampled. Implementing this procedure will address the need for simple and reliable collection and application of MSC-EVs in translational research.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% povidone-iodine (Betadine)</td>
			<td>Weizhenyuan</td>
			<td>10053956954292</td>
			<td>Wound disinfection</td>
		</tr>
		<tr>
			<td>Calibration solution</td>
			<td>Particle Metrix</td>
			<td>110-0020</td>
			<td>Calibrate the NTA instrument</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Sigma</td>
			<td>53716-49-7</td>
			<td>Analgesic medicine</td>
		</tr>
		<tr>
			<td>Caudal vein imager&#160;</td>
			<td>KEW Life Science</td>
			<td>KW-XXY</td>
			<td>Caudal vein imager</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5418R</td>
			<td>Centrifugation</td>
		</tr>
		<tr>
			<td>Fatal bovine serum</td>
			<td>Corning</td>
			<td>35-081-CV</td>
			<td>Culture of UCMSCs</td>
		</tr>
		<tr>
			<td>Formvar/carbon-coated square mesh</td>
			<td>PBL Assay Science&#160;</td>
			<td>24916-25</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Zhongke Life Science</td>
			<td>Z8G5JBMz</td>
			<td>Post-treatment care of animals</td>
		</tr>
		<tr>
			<td>Heparin Solution</td>
			<td>StemCell</td>
			<td>7980</td>
			<td>Systemic injection</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD Life Science</td>
			<td>R510-22</td>
			<td>Animal anesthesia</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium Alpha basic (1x)</td>
			<td>Gibco</td>
			<td>C12571500BT</td>
			<td>Culture of UCMSCs</td>
		</tr>
		<tr>
			<td>Nanoparticle tracking analyzer</td>
			<td>Particle Metrix</td>
			<td>ZetaView PMX120</td>
			<td>Nanoparticle tracking analysis</td>
		</tr>
		<tr>
			<td>PBS (1x)</td>
			<td>Meilunbio</td>
			<td>MA0015</td>
			<td>Resuspend EVs</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Procell Life Science</td>
			<td>PB180120</td>
			<td>Culture of UCMSCs</td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td>Solarbio</td>
			<td>12501-23-4</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf</td>
			<td>3120000224</td>
			<td></td>
		</tr>
		<tr>
			<td>PKH26 Red Fluorescent Cell Linker Kit</td>
			<td>Sigma-Aldrich</td>
			<td>MINI26</td>
			<td>Labeling EVs</td>
		</tr>
		<tr>
			<td>Skin biopsy punch</td>
			<td>Acuderm</td>
			<td>69038-10-50</td>
			<td>Skin defects</td>
		</tr>
		<tr>
			<td>Software ZetaView</td>
			<td>Particle Metrix</td>
			<td>Version 8.05.14 SP7&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic equipment</td>
			<td>Grant</td>
			<td>v-0001-0005</td>
			<td>Water bath</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>HITACHI</td>
			<td>HT7800</td>
			<td>Transmission electron microscope</td>
		</tr>
		<tr>
			<td>UCMSCs</td>
			<td>Bai&#39;ao&#160;</td>
			<td>UKK220201</td>
			<td>Commercially UCMSCs</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman</td>
			<td>XPN-100</td>
			<td>Centrifugation</td>
		</tr>
		<tr>
			<td>Ultrapure filtered water purification system</td>
			<td>Milli-Q</td>
			<td>IQ 7000</td>
			<td>Preparation of ultrapure water</td>
		</tr>
	</tbody>
</table>

isolation, characterization, and therapeutic application of extracellular vesicles from cultured human mesenchymal stem cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

MVs and Exos from cultured human UCMSCs are isolated following the experimental workflow (Figure 1). The NTA results demonstrate that the size of Exos from human MSCs ranges from 40 nm to 335 nm with a peak size of about 100 nm, and the size of MVs ranges from 50 nm to 445 nm with a peak size of 150 nm (Figure 2). Morphological characterization of MSC-derived Exos exhibit a typical cup shape (Figure 3). EVs are efficiently labeled b...

Watch this Scientific Journal Video about Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells at JoVE.com

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

This procedure addressed the need for simple and reliable mesenchymal stem cell-derived extracellular vesicle collection, and of application in translation of research. Collection of extracellular vesicles by differential centrifugation is simple, readily scalable, and reproducible, which will be useful to the field. Extracellular vesicles indistinct}from indistinct}mesenchymal stem cell is a indistinct}application in modern disease treatments.I will not be afraid of mistakes. This operation is simple and easy to learn. There are many details in this simple program that can be better presented through video.To begin, culture the mesenchymal stem cells to more than 90%confluence. Replace the culture medium in each dish with 8 mL of supplemented alpha-MEM. After 48 hours of culture, the cells must be grown evenly to fully collect the culture medium into 50 mL centrifuge tubes.Then, centrifuge the culture medium at 800 x g for 10 minutes at four degrees Celsius. Transfer the supernatant to 1.5 mL conical tubes to remove the cell fragments and cellular debris. Centrifuge the supernatant at 16, 000 x g for 30 minutes at four degrees Celsius.Use a pipette to transfer the suspension to ultracentrifuge tubes. Then, centrifuge at 150, 000 x g for two hours at four degrees Celsius. Resuspend the microvesicle pellet in 50 uL of PBS per dish.Discard the supernatant and collect the residue, which is exosomes. Resuspend the exosomes from seven dishes of cells in 50 uL of PBS. In a 15 mL tube, dilute 1 uL of extracellular vesicles in 1, 499 uL of PBS and mix.Then, start the compatible software for the tracking analyzer. Fill the sample cell with distilled water and then allow the instrument to start the cell check. Calibrate the instrument with a prepared standard solution.Ensure that the particle number displayed on the software detection interface is between 50 to 400, preferably around 200, and click okay. Next, rinse the sample cell with 5 mL of distilled water. Before sample analysis, flush the channel with 1 mL of distilled water.Ensure that the particle number displayed on the software detection interface is less than 10. In a 1.5 mL conical tube, resuspend the extracellular vesicles with 250 uL of PBS. In another 1.5 mL conical tube, prepare the working solution of the PKH26 dye.Mix the resuspended extracellular vesicles with the working solution. Allow it to stand at room temperature for five minutes and then add 500 uL of EV-depleted FBS to stop the reaction. For microvesicles, centrifuge at 16, 000 x g for 30 minutes at four degrees Celsius.Discard the supernatant. Add 1 mL of PBS to rinse the residue. Centrifuge again, and discard the supernatant to remove the unbound dye.For microvesicles, centrifuge at 16, 000 x g for 30 minutes at four degrees Celsius. Resuspend the residue with 200 uL of PBS. Drop 20 uL of suspension on the slide and observe it under a fluorescence microscope.Resuspend extracellular vesicles in 200 uL of PBS, and then mix with heparin solution at a 10:1 volume ratio. Place the mouse into the caudal vein imaging system. Press the switch to lift the lever.With the help of a tail vein illustrator, perform a systematic injection of the extracellular vesicle suspension through the tail vein. Analysis of particle size distribution using NTA demonstrated that the size of exosomes from human mesenchymal stem cells ranged from 40 to 335 nm, with a peak size of about 100 nm. Also, the size of microvesicles ranged from 50 to 445 nm, with a peak size of 150 nm.Morphological characterization of exosomes showed that they exhibited a typical cup shape. Extracellular vesicles were efficiently labeled by PKH26, which was observed by the gross view as labeled pellets, as well as by fluorescent microscopy. The operator should pay attention during supernatant collection, because cells must be blown evenly to fully collect the released EVs retained in the indistinct}of mesenchymal stem cells.In order to verify the success of the injection, in vivo imaging of biodistribution of fluorescence-labeled EVs or frozen sections of specific tissue sites of engraftment can be performed.

isolation-characterization-therapeutic-application-extracellular

Research

JoVE Journal

Biology

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation &amp; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential biotechnology models. Recent work has demonstrated that both marine and freshwater cyanobacteria produce extracellular vesicles -&#160;small membrane-bound structures released from the outer surface of the microbes. While vesicles likely contribute to diverse biological processes, their specific functional roles in cyanobacterial biology remain largely unknown. To encourage and advance research in this area, a detailed protocol is presented for isolating, concentrating, and purifying cyanobacterial extracellular vesicles. The current work discusses methodologies that have successfully isolated vesicles from large cultures of Prochlorococcus, Synechococcus, and Synechocystis. Methods for quantifying and characterizing vesicle samples from these strains are presented. Approaches for isolating vesicles from aquatic field samples are also described. Finally, typical challenges encountered with cyanobacterial vesicle purification, methodological considerations for different downstream applications, and the trade-offs between approaches are also discussed.

The present protocol providesdetailed descriptions for isolation, concentration,and characterization of extracellular vesicles from cyanobacterial cultures. Approaches for purifying vesicles from cultures at different scales, trade-offs among methodologies, and considerations for working with field samples are also discussed.