Name: formulating and characterizing an exosome-based dopamine carrier system
Uploaded: 2022-04-04T14:00:00
Description: Watch this Scientific Journal Video about Formulating and Characterizing an Exosome-based Dopamine Carrier System at JoVE.com

The work was primarily supported by research funding provided by Y&#305;ld&#305;z Technical University Scientific Research Projects (TSA-2021-4713).

NOTE: See the Table of Materials for details related to all materials and equipment used in this protocol.
1. Culturing and cryopreservation of Wharton&#39;s jelly mesenchymal stem cells
<ol>
	<li>Remove the WJ-MSCs (from ATCC) from the -80 &#176;C freezer. Seed the cells into a flask containing DMEM-F12 medium supplemented with 10% fetal bovine serum (FBS). Incubate them at 37 &#176;C in an incubator containing 5% CO2.</li>
	<li>Change the cultur...

Exosomes are small membrane vesicles with dimensions of 40-200 nm secreted by most cell types, e.g., MSCs1. Capable of enabling communication between cells, exosomes can enter cells in different ways such as endocytosis, phagocytosis, micropinocytosis, lipid-mediated internalization, and fusion33,44. Compared to other nanocarrier systems, the lipids and cholesterol found on the exosome surface confer the ability to carry both hydrophilic a...

Exosomes, bioactive vesicles with significant features, range in size from 40 nm to 200 nm. Exosomes originate from the cell membrane and are formed because of the release of the endosomes1. These structures serve as cell-to-cell communicators and interact with neighboring cells to facilitate the transfer of active molecules. Exosomes can be isolated from many different sources. These include body fluids such as plasma, urine, cerebrospinal fluid, saliva, as well as cell lines cultured under in vitro conditions. Exosomes have an important role in the elimination of nerve damage, thanks to the biomacromolecules they contain, such as lipids, proteins, and nucleic acids2. Glia, which are the supporting cells of the nervous system3, transfer proteins and micro RNAs to the axons of neurons via exosomes4.
Lipids forming the myelin sheath, which are a characteristic feature in nerve conduction, are also released from oligodendrocytes via exosomes4,5. Exosomes are also involved in processes such as synaptic plasticity, neuronal stress response, cell-cell communication, and neurogenesis in the brain6,7. The fact that exosomes possess nano-dimensions enables them to pass through the BBB. There is a special transition route from the interstitial fluid to the cerebrospinal fluid after penetrating this membrane8. Thanks to their surface properties, exosomes can interact efficiently with target cells as a drug delivery system and actively deliver the loaded drugs.
Due to the expression of various adhesive proteins (tetraspanins and integrins) on the surface of exosomes, these extracellular vesicles can easily interact and fuse with host cell membranes9. It is thought that exosomes can be used as a drug delivery system, especially in the treatment of central nervous system diseases due to their ability to penetrate the BBB and their surface properties. Mesenchymal stem cell (MSC)-derived exosomes have a lower risk of immune rejection compared to allogeneic cellular therapies, and in this respect, they can be an important component of cell-free treatment applications10.
Dopamine is a molecule whose deficiency in the brain is the characteristic feature of Parkinson&#39;s disease (PD), worsening day by day11,12,13. It is known that PD is associated with degeneration of dopaminergic neurons in the substantia nigra of the mesencephalon and loss of motor neuron functions14,15. The death of dopaminergic neurons prevents the supply of the neurotransmitter dopamine to the brain striatum. This, in turn, results in the emergence of PD-specific symptoms16. These symptoms of PD are bradykinesia, postural instability, rigidity, and especially resting tremor12,13. Although PD was first described more than two centuries ago, studies to understand the pathology and etiology of the disease are still ongoing and it is currently accepted that PD is a complex systemic disease17. It is predicted that dopamine deficiency occurs, and clinical PD symptoms are observed when more than 80% of neurons degenerate18. In the treatment of the disease, incomplete dopamine supplementation is preferred to reduce motor symptoms. In vivo studies have shown that direct infusion of dopamine into the brain significantly reduces symptoms in animals19. Dopamine precursors such as L-DOPA (L-3,4-dihydroxyphenylalanine) and dopamine receptor drugs are used in the clinic because the direct infusion of dopamine into the brain is not possible in humans and dopamine entering the system cannot cross the BBB20. These types of drugs lose their effectiveness over time. However, there is still no curative treatment approach for PD. Hence, there is a huge necessity to develop new therapeutic strategies and treatment modalities to reveal the pathophysiology of the disease and reduce the impact of PD on patients.
Exosome-based studies have recently attracted attention for gathering information about both therapeutic approaches and pathologies of nervous system diseases. MSC-derived exosomes have been shown to reduce inflammation in nerve damage and contribute to neuronal regeneration21,22,23. In addition, it has been reported that MSC-derived exosome secretomes reduce apoptosis by showing neurotrophic and neuroprotective effects, especially on dopaminergic neurons24,25. Research on platforms in which exosomes are used as therapeutic drug delivery systems have intensively accelerated in recent years. In numerous studies, it has been observed that relevant drugs can be easily encapsulated into exosomes and delivered safely into target cells, tissues, and organs26,27. Different methods such as incubation, freeze/thaw cycles, sonication, and extrusion could be used for drug loading into exosomes28.
Coincubation with exosomes or exosome-like vesicles allows lipophilic small molecules to be passively encapsulated into these delivery systems28,29,30. In particular, various molecules such as curcumin31, catalase30, doxorubicin32, and paclitaxel33&#160;were effectively loaded into exosomes. It has been observed that catalase-containing exosomes, which have antioxidant activity, efficiently accumulated in the neurons and microglial cells in the brain and exhibited strong neuroprotective activities30. In the same study, saponin, added into the complex to increase the loading efficiency, was found to increase the drug loading percentage during incubation30,34. However, further studies are needed to establish the standards for drug loading into exosomes.
This paper describes the development of a nanocarrier system by encapsulation of dopamine into exosomes that were isolated from WJ-MSCs. All steps, including the cultivation of WJ-MSCs,&#160;isolation, and characterization of exosomes, drug loading experiments, characterization of dopamine-loaded exosomes with various techniques, and in vitro cytotoxicity analysis are explained in detail.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m membrane filter</td>
			<td>Aisimo</td>
			<td></td>
			<td>Used for the sterilization process</td>
		</tr>
		<tr>
			<td>0.45 &#181;m syringe filter</td>
			<td>Aisimo</td>
			<td></td>
			<td>Used for the sterilization process</td>
		</tr>
		<tr>
			<td>15 mL Falcon tube</td>
			<td>Nest</td>
			<td></td>
			<td>Used in cell culture step</td>
		</tr>
		<tr>
			<td>25 cm2 cell culture flasks (Falcon, TPP tissue culture flasks</td>
			<td>Nest</td>
			<td></td>
			<td>Used in cell culture step</td>
		</tr>
		<tr>
			<td>50 mL Falcon tube</td>
			<td>Nest</td>
			<td></td>
			<td>Used in cell culture step</td>
		</tr>
		<tr>
			<td>75 cm2 cell culture flasks (Falcon, TPP tissue culture flasks</td>
			<td>Nest</td>
			<td></td>
			<td>Used in cell culture step</td>
		</tr>
		<tr>
			<td>96 well plates (Falcon, TPP microplates)</td>
			<td>Merck Millipore</td>
			<td></td>
			<td>Used in cell culture step</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma</td>
			<td>271004-1L</td>
			<td>Used for HPLC analysis</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>NUVE-OT 90L</td>
			<td></td>
			<td>Used for the sterilization process</td>
		</tr>
		<tr>
			<td>Cell Culture Cabin</td>
			<td>Hera Safe KS</td>
			<td></td>
			<td>Used for the cell culture process</td>
		</tr>
		<tr>
			<td>Centrifugal</td>
			<td>Hitachi</td>
			<td>CF16RN</td>
			<td>Used in the exosome isolation step</td>
		</tr>
		<tr>
			<td>CO2 incubator with Safe Cell UV</td>
			<td>Panasonic</td>
			<td></td>
			<td>Used for the cell culture process</td>
		</tr>
		<tr>
			<td>Dopamine hydrochloride H8502-10G</td>
			<td>Sigma</td>
			<td>H8502-10G</td>
			<td>Used in exosome dopamine loading</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium/Nutrient Mixture-F12</td>
			<td>Sigma</td>
			<td>RNBJ7249</td>
			<td>Used as cell culture medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum-FBS</td>
			<td>Capricorn</td>
			<td>FBS-16A</td>
			<td>It was used by adding to the cell culture medium.</td>
		</tr>
		<tr>
			<td>Freezer -80 &#176;C</td>
			<td>Panasonic</td>
			<td>MDF-U5386S-PE</td>
			<td>To store cells and the resulting exosomes</td>
		</tr>
		<tr>
			<td>Fridge</td>
			<td>Panasonic</td>
			<td>MPR-215-PE</td>
			<td>Used to store cell culture and other materials</td>
		</tr>
		<tr>
			<td>High performance liquid chromatography-HPLC</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td>The presence of dopamine from the content of the obtained formulation was investigated.</td>
		</tr>
		<tr>
			<td>Microscope- Primovert</td>
			<td>Zeiss</td>
			<td></td>
			<td>Used to observe cells in cell culture.</td>
		</tr>
		<tr>
			<td>MTT Assay</td>
			<td>Biomatik</td>
			<td></td>
			<td>Used to measure cell viability</td>
		</tr>
		<tr>
			<td>NanoSight NS300</td>
			<td>Malvern panalytical</td>
			<td>Malvern panalytical</td>
			<td>Used for exosome characterization</td>
		</tr>
		<tr>
			<td>Optima XPN-100 Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Used in the exosome isolation step</td>
		</tr>
		<tr>
			<td>PBS tablet</td>
			<td>Biomatik</td>
			<td>43602</td>
			<td>In the preparation of the PBS solution</td>
		</tr>
		<tr>
			<td>Penicilin/Streptomycin Solution</td>
			<td>Capricorn</td>
			<td>PB-S</td>
			<td>It was added to the medium to prevent contamination in cell culture.</td>
		</tr>
		<tr>
			<td>Pipette Aid</td>
			<td>Isolab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Precision balance-Kern</td>
			<td>Kern-ABJ220-4NM</td>
			<td></td>
			<td>Used in the preparation of solutions</td>
		</tr>
		<tr>
			<td>Q500 Sonicator</td>
			<td>Qsonica, LLC</td>
			<td></td>
			<td>Used to digest exosomes in HPLC analysis</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma</td>
			<td>47036-50G-F</td>
			<td>It was used by adding it to the total solution in the exosome dopamine loading process.</td>
		</tr>
		<tr>
			<td>Spectrostar-Nano-Spectrophotometry</td>
			<td>BMG LABTECH</td>
			<td></td>
			<td>Used for MTT and drug release analyzes</td>
		</tr>
		<tr>
			<td>SPSS 22</td>
			<td></td>
			<td></td>
			<td>statistical package program</td>
		</tr>
		<tr>
			<td>Vorteks-FinePCR</td>
			<td>FinePCR-FineVortex</td>
			<td></td>
			<td>Used to mix solutions homogeneously</td>
		</tr>
		<tr>
			<td>Water Bath 37 &#176;C-Senova</td>
			<td>Senova</td>
			<td></td>
			<td>Used in cell culture step</td>
		</tr>
		<tr>
			<td>Wharton&#8217;s jelly mesenchymal stem cells</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ZetaSizer</td>
			<td>Malvern Nano ZS</td>
			<td>Malvern Nano ZS</td>
			<td>Used for exosome characterization</td>
		</tr>
	</tbody>
</table>

formulating and characterizing an exosome-based dopamine carrier system

Exosomes between 40 and 200 nm in size constitute the smallest subgroup of extracellular vesicles. These bioactive vesicles secreted by cells play an active role in intercellular cargo and communication. Exosomes are mostly found in body fluids such as plasma, cerebrospinal fluid, urine, saliva, amniotic fluid, colostrum, breast milk, joint fluid, semen, and pleural acid. Considering the size of exosomes, it is thought that they may play an important role in central nervous system diseases because they can pass through the blood-brain barrier (BBB). Hence, this study aimed to develop an exosome-based nanocarrier system by encapsulating dopamine into exosomes isolated from Wharton's jelly mesenchymal stem cells (WJ-MSCs). Exosomes that passed the characterization process were incubated with dopamine. The dopamine-loaded exosomes were recharacterized at the end of incubation. Dopamine-loaded exosomes were investigated in drug release and cytotoxicity assays. The results showed that dopamine could be successfully encapsulated within the exosomes and that the dopamine-loaded exosomes did not affect fibroblast viability.

Exosome isolation and characterization 
Wharton jelly stem cells are cultured and incubated in a serum-free medium for 48 h when the culture reaches sufficient density. After the end of the incubation, the supernatant is stored at -20 &#176;C. The collected supernatants are diluted with PBS and subjected to ultracentrifugation (Figure 1). The obtained solution is analyzed by NTA and DLS analyses. The exosomes are sterilized by passing through a 0.22 &#181;m filter. The ...

Watch this Scientific Journal Video about Formulating and Characterizing an Exosome-based Dopamine Carrier System at JoVE.com

Formulating and Characterizing an Exosome-based Dopamine Carrier System

Here, we aimed to obtain a formulation by dopamine loading of the isolated exosomes of stem cells from Wharton's jelly mesenchymal stem cells. Exosome isolation and characterization, drug loading into the resulting exosomes, and the cytotoxic activity of the developed formulation are described in this protocol.

Exosomes between 40 and 200 nm in size constitute the smallest subgroup of extracellular vesicles. These bioactive vesicles secreted by cells play an ...

Exosomes might react to vesicles secreted by a cell between 14 and 200 nanometer in size, and split from in the intracellular cargo and communication, which comes to the smallest subgroup of extracellular vesicles. Exosomes which are formed as a result of the differentiation of the endosome, originate from the cell membrane. The stem cells can be isolated from different sources such as bone marrow or adipose tissues.but adult tissue dried stem cells have higher immunogenic potential Cons springing the size of exosomes, it's told that it's made an important role in central nervous system disease because it can pass through to the blood brain barrier. To begin, wash the seeded Wharton's jelly mesenchymal stem cells with five milliliters of PBS. Then add five milliliters of trypsin EDTA solution before placing the flask in an incubator.After incubation, collect the cells and centrifuge at 1, 500 G for five minutes. Then remove the supernatant and add one milliliter of DMEM-F12 containing 10%FBS to the tube. Wash cultured cells with PBS.After washing, add serum free DMEM-F12 medium to the cells and place it in the incubator. To isolate the exosomes, collect the serum free media and centrifuge at 300 G at four degrees Celsius for 10 minutes. Transfer the supernatant to another tube and process it by serial centrifugation at higher speeds.After the final centrifugation, slowly remove the supernatant and dissolve the pellet in one milliliter PBS. Mix by vortexing and proceed with ultracentrifugation at 110, 000 G for 70 minutes at four degrees Celsius. Add three milliliters of PBS to the exosome suspension collected earlier by ultracentrifugation and sterilize the diluted suspension by filtration using a 0.22 micron filter.Then transfer 500 microliters of the sterilized exosome suspension to another tube. Next sequentially, add 500 microliters of 0.5 milligrams per milliliter dopamine solution and saponin into the sterilized suspension. After incubation ultracentrifuge the suspension.The sample can either be used for NTA and DLS analysis to characterize dopamine loaded exosomes or stored at minus 20 degrees Celsius until further use. In the NTA analysis, the average size of Wharton jelly derived exosomes was determined to be 98 nanometers and dopamine loaded exosomes to be 110 nanometers. The numbers of nanoparticles revealed by NTA and DLS analyses were consistent with each other.The zeta potential of Wharton jelly derived exosomes was measured as minus 15.7 and dopamine loaded exosomes as minus 17.6 millivolts. The HPLC analysis of the dopamine loaded exosomes detected the peak for dopamine at 6.45 minutes. The cumulative drug release profile revealed that 74.8%of encapsulated dopamine was released from the exosomes within the first eight hours.The cytotoxicity of the dopamine loaded and free exosomes on fibroblasts was investigated. Although the dopamine loaded exosomes did not demonstrate any cytotoxic effects, saponin decreased the fibroblast viability. The MTT assay showed increased cell viability when the fibroblasts were treated with the dopamine loaded exosomes.The statistical significance of the results was evaluated by performing one-way anova. Additionally, the cytotoxic effects of different concentrations of free exosomes were investigated on the fibroblasts. The fibroblast viability was higher with free exosomes at the concentration of 25 microliters per milliliter.It's really important for the central nervous system is the patient sizes, the play role in the processes such as plasticity, neuro response, side to side communication, and neurogenesis in the brain. In addition, thanks to surface of exosomes, it says, has been observed that they can in interact with with target cell drug and activity delivered during the drug Is a completion. For the first time in the study, we developed a new drug formulation by encapsulating dopamine into stem cell derived exosomes.He proposed that this formulation will be so promising for treatment of Parkinson's disease.

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Bioengineering

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.

Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various biological activities including cell proliferation, survival, migration and differentiation. Several aberrations in the FGFR signaling pathway, owing to mutations or gene amplification events, have been identified in various types of cancers. Hence, recent research has focused on developing strategies involving therapeutic targeting of FGFRs. Current FGFR inhibitors at various stages of pre-clinical and clinical development include either small molecule inhibitors of tyrosine kinases or monoclonal antibodies, with only a few peptide- based inhibitors in the pipeline. Here, we provide a protocol using phage display technology to screen small peptides as antagonists of FGFR2. Briefly, a library of phage-displayed peptides was incubated in a plate coated with FGFR2. Subsequently, unbound phage was washed off by TBST (TBS + 0.1% [v/v] Tween-20), and bound phage was eluted with 0.2 M glycine-HCl buffer (pH 2.2). The eluted phage was further amplified and used as input for the next round of biopanning. Following three rounds of biopanning, the peptide sequences of individual phage clones were identified by DNA sequencing. Finally, the screened peptides were synthesized and analyzed for affinity and biological activity.

Herein, we present a detailed protocol for screening small peptides that bind to FGFR2 using a phage display peptide library. We further analyze the affinity of the selected peptides toward FGFR2 in vitro and its ability to suppress cell proliferation.

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...