Name: Forfatter Spotlight: Fremskridt inden for nanopartikelteknologi til lægemiddellevering og immunterapi
Uploaded: 2023-11-10T12:50:00
Description: Watch this Scientific Journal Video about Mechanical Dissociation of Tissues for Single Cell Analysis Using a Motorized Device at JoVE.com

Finansiering blev modtaget fra Fischell Department of Bioengineering (KM), T32 GM080201 (MA), Vogel Endowed Summer Fellowship (MA), LAM Foundation (KM) og American Lung Association (KM). Forfatterne ønsker at takke Michele Kaluzienski for hjælp med redigering.

Denne protokol blev godkendt af UMD Institutional Animal Care and Use Committee (IACUC). Væv fra 7 til 9 uger gamle C57BL/6J-hunmus blev anvendt til disse undersøgelser. Dyrene blev hentet fra en kommerciel kilde (se materialetabel).
1. Manuel afkobling
BEMÆRK: Dette trin er tilpasset fra Maisel K. et al.7.
<ol><li>Fjern dissekeret væv og læg det i koldcellekulturmedier med 5% føtalt bovint serum (FBS...

Semi-automatiseret mekanisk-enzymatisk vævsdissociation til enkeltcelleanalyse

<ol>
	<li>Wiegleb, G., Reinhardt, S., Dahl, A., Posnien, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+dissociation+for+single-cell+and+single-nuclei+RNA+sequencing+for+low+amounts+of+input+material.">Tissue dissociation for single-cell and single-nuclei RNA sequencing for low amounts of input material.</a> Frontiers in Zoology. 19 (1), 27 (2022).</li><li>Weiskirchen, S., Tag, C. G., Sauer-Lehnen, S., Tacke, F., Weiskirchen, R., Ritti&#233;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+primary+murine+hepatic+stellate+cells.">Isolation and culture of primary murine hepatic stellate cells.</a> Fibrosis: Methods and Protocols. , 7113-7118 (2017).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+and+characterization+of+endothelial+cells+from+juvenile+nasopharyngeal+angiofibroma.">The isolation and characterization of endothelial cells from juvenile nasopharyngeal angiofibroma.</a> Acta Biochimica et Biophysica Sinica. 48 (9), 856-858 (2016).</li><li>Burja, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+tissue+dissociation+protocol+for+single-cell+rna+sequencing+analysis+of+fresh+and+cultured+human+skin+biopsies.">An optimized tissue dissociation protocol for single-cell rna sequencing analysis of fresh and cultured human skin biopsies.</a> Frontiers in Cell and Developmental Biology. 10, 102022 (2022).</li><li>McBeth, C., Gutermuth, A., Ochs, J., Sharon, A., Sauer-Budge, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+tissue+dissociation+for+rapid+extraction+of+viable+cells.">Automated tissue dissociation for rapid extraction of viable cells.</a> Procedia CIRP. 65, 88-92 (2017).</li><li>Welch, E. C., Yu, H., Barabino, G., Tapinos, N., Tripathi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric-field+facilitated+rapid+and+efficient+dissociation+of+tissues+into+viable+single+cells.">Electric-field facilitated rapid and efficient dissociation of tissues into viable single cells.</a> Scientific Reports. 12 (1), 10728 (2022).</li><li>Maisel, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-lymphangiogenic+VEGFR-3+signaling+modulates+memory+T+cell+responses+in+allergic+airway+inflammation.">Pro-lymphangiogenic VEGFR-3 signaling modulates memory T cell responses in allergic airway inflammation.</a> Mucosal Immunology. 14 (1), 144-151 (2021).</li><li>Karmakar, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pilot+study+to+determine+the+utility+of+automated+tissue+dissociator+for+flowcytometry+based+evaluation+of+hematolymphoid+tumor+tissue+biopsies.">A pilot study to determine the utility of automated tissue dissociator for flowcytometry based evaluation of hematolymphoid tumor tissue biopsies.</a> Indian Journal of Hematology and Blood Transfusion. 38 (2), 403-410 (2022).</li><li>Montanari, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated-mechanical+procedure+compared+to+gentle+enzymatic+tissue+dissociation+in+cell+function+studies.">Automated-mechanical procedure compared to gentle enzymatic tissue dissociation in cell function studies.</a> Biomolecules. 12 (5), 701 (2022).</li><li>Genova, A., Dix, O., Saefan, A., Thakur, M., Hassan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carpal+tunnel+syndrome:+a+review+of+literature.">Carpal tunnel syndrome: a review of literature.</a> Cureus. 12 (3), e7333 (2020).</li></ol>

Denne enhed er designet til nem montering i forskningsindstillingen for at tilvejebringe enkeltcellesuspensioner fra hele væv til efterfølgende enkeltcelleanalyse. Funktionerne, selvom de er grundlæggende, er tilstrækkelige til at imødekomme forskernes behov i akademiske omgivelser og videre. En vigtig fordel ved at bruge denne enhed er dens potentiale til at forbedre forberedelsen af enkeltcellesuspensioner ved at reducere variabiliteten. Derudover kan evnen til at behandle 12+ prøver samtidigt forbedre konsistens...

Enkeltcelleanalyse er hurtigt blevet afgørende for nye biomedicinske opdagelser, hvad enten det drejer sig om applikationer som flowcytometri, identifikation af forskellige celletyper, enkeltcellesekventering eller til identifikation af genomiske eller transkriptomiske variationer mellem celler1. Udførelse af sådanne celleisoleringer fra væv af interesse kræver hakning af dissekeret væv og skubbe dem gennem en fincellesil for at filtrere bindevæv fra de ønskede celler (figur 1A). Isolering af klæbende celletyper, såsom dendritiske celler eller makrofager eller celler fra særligt fibrøst væv, kræver yderligere mekaniske eller enzymatiske separationstrin 2,3,4. Denne proces udføres generelt manuelt, hvilket gør den meget tidskrævende og tilbøjelig til brugervariabilitet ved vurdering af celleudbytter og prøvelevedygtighed. Derfor er det afgørende at introducere tilpassede muligheder for automatiseret vævsdissociation. Selv om der er gjort visse forsøg på at designe sådanne systemer, er de eksisterende muligheder ikke altid let tilgængelige, især i akademiske laboratorier og miljøer med lavere ressourcer, hovedsagelig på grund af disse enheders omkostningsuoverkommelige karakter5. Desuden kan disse enheder ikke altid tilpasses til de individuelle behov i en forskningsgruppe6.
Her blev en vævsdissociatoranordning designet til at automatisere fordøjelsen af hele væv eller vævsstykker i enkeltcellesuspensioner ved hjælp af fordøjelsesenzymer og mekanisk forstyrrelse. Denne enhed kan let samles i laboratoriet, placeres i varme- eller kølekamre til temperaturregulering, tilpasses til det krævede antal væv til dissociering og programmeres med de ønskede dissociationsprotokoller. Den brede anvendelse af denne enhed kunne forbedre reproducerbarheden af celleekstraktionsprotokoller betydeligt og give et tidsbesparende alternativ til manuel dissociation.
Designet giver mulighed for samtidig fordøjelse af op til 12 væv gennem en automatiseret proces. Enheden består af 12 individuelle motorer, der er forbundet parallelt og drives af et standard vægstik gennem en AC / DC-adapter med et justerbart spændingshjul til styring af motorernes rotation / hastighed. Motorerne drejer en unbrakobolt, der passer tæt ind i toppen af C-rørene. C-rørene holdes på plads ved nedadgående spænding på en akrylplade, der låser på hver side til toppladen, hvor motorerne er fastgjort (figur 1B). Fordi motorerne er forbundet parallelt, bør deres hastighed ved en given spænding ikke variere meget, men belastningen (antallet af C-rør monteret på enheden) vil påvirke hastigheden, selv når spændingen holdes konstant. For at måle rotationer pr. minut (omdr./min.) er der indbygget et omdrejningstæller ved hjælp af en hall-effektsensor og en fast magnet på en af motorakslerne (supplerende figur 1). CAD-filerne til opbygning af motorarrays findes i supplerende kodningsfil 1. Der medfølger også en programmerbar kontakt til at vende rotationsretningen ved at vende de positive/negative ladninger, der leveres til motorerne. Alle disse funktioner er integreret ved hjælp af kodet software (Arduino IDE-software, se materialetabel) på en Arduino Nano (supplerende kodningsfil 2). Ved hjælp af tilsluttede knapper og et LCD-panel (supplerende figur 2) er det muligt at oprette og køre gemte og brugerdefinerede protokoller, automatisk vende rotationsretningen på bestemte tidspunkter af en protokol, justere hastigheden ved hjælp af spændingen (supplerende figur 3) og vise den aktuelle motorhastighed og den tid, der er tilbage til at fuldføre en programmeret protokol (supplerende figur 4).
Til denne undersøgelse blev enkeltcellesuspensioner fremstillet under anvendelse af både mekanisk-enzymatisk vævsdissociation med denne enhed og manuel-enzymatisk vævsdissociation for at bestemme eventuelle forskelle i celler, der blev genfundet til downstream-applikationer. Cellepræparaterne blev evalueret baseret på samlede celleudbytter pr. væv og procent cellelevedygtighed. Flowcytometri blev brugt til at sammenligne potentielle forskelle i overflademarkørekspression. Data blev analyseret ved hjælp af graftegning og statistisk analyse software. Uparrede Welch t-tests blev brugt til at sammenligne par af prøver eller grupper, hvor prøvestørrelser n > 4 mus repræsenterede 2 replikate eksperimenter. Detaljerede instruktioner til fremstilling og samling af denne enhed findes i supplerende fil 1. Materialer, der er nødvendige til denne protokol, er angivet i materialetabellen.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#188; inch acrylic sheet&#160; 12&#34; x 24&#34;</td>
			<td>Acrylic Mega Store</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>&#189; inch acrylic sheet 12&#34; x 12&#34;</td>
			<td>SimbaLux</td>
			<td>SL-AS13-12x12</td>
			<td></td>
		</tr>
		<tr>
			<td>12 G stainless steel wire (for tension arms)</td>
			<td>Everbilt</td>
			<td>1000847413</td>
			<td></td>
		</tr>
		<tr>
			<td>16 G electrical wire (stranded)</td>
			<td>Best Connections</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>2 x 3 mm magnet&#160;&#160;</td>
			<td>SU-CRO0587</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>2-channel relay board (to reverse polarity of current to motors)</td>
			<td>AEDIKO</td>
			<td>AE06233</td>
			<td></td>
		</tr>
		<tr>
			<td>37 mm Diameter DC Motors (12 V, 200 rpm) x 12</td>
			<td>Greartisan</td>
			<td>N/A</td>
			<td>Rated Torque: 2.2 Kg.cm 
			Reduction Ratio: 1:22 
			Rated Current: 0.1 A 
			D Shaped Output Shaft Size: 6 x 14mm (0.24&#34; x 0.55&#34;) (D x L) 
			Gearbox Size: 37 x 25 mm (1.46&#34; x 0.98&#34;) (D x L) 
			Motor Size: 36.2 x 33.3 mm (1.43&#34; x 1.31&#34;) (D x L) 
			Mounting Hole Size: M3 (not included)</td>
		</tr>
		<tr>
			<td>AC/C Power Adapter with variable voltage controller&#160;&#160; (5 Amps, 3-12 volts)</td>
			<td>Mo-gu</td>
			<td>J19091-2-MG-US</td>
			<td></td>
		</tr>
		<tr>
			<td>AC-DC 5 V 1 A Precision buck converter step down transformer</td>
			<td>Walfront</td>
			<td>1A</td>
			<td>(power adapter for powering Arduino Nano)</td>
		</tr>
		<tr>
			<td>Arduino Nano&#160;&#160; (Lafvin)</td>
			<td>LAFVIN</td>
			<td>8541582500</td>
			<td></td>
		</tr>
		<tr>
			<td>Buttons&#160;</td>
			<td>Awpeye</td>
			<td>Push-button</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL6/J mice&#160;</td>
			<td>Jackson Laboratory</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase 4</td>
			<td>Worthington</td>
			<td>CLS4 LS004188</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Roche</td>
			<td>11088866001</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Roche</td>
			<td>11284932001</td>
			<td></td>
		</tr>
		<tr>
			<td>Double sided foam tape</td>
			<td>SANKA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Double Sided prototyping circuit board</td>
			<td>deyue</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma- Aldrich</td>
			<td>E7889</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrical solder and soldering iron</td>
			<td>LDK</td>
			<td>1002P</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrical Tape</td>
			<td>3M</td>
			<td>03429NA</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS (Fetal Bovine Serum)</td>
			<td>Gibco</td>
			<td>16140089</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS C Tubes</td>
			<td>Miltenyi</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad Prism</td>
			<td>GraphPad, La Jolla, CA</td>
			<td></td>
			<td>Graphing and statistical analysis software</td>
		</tr>
		<tr>
			<td>Hall effect sensor Dimensions : 0.79 x 0.79x 0.39 inches</td>
			<td>SunFounder</td>
			<td>43237-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Hex Coupler 6 mm Bore Motor Brass x 2 x 12</td>
			<td>Uxcell</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hex head bolts (M4-.70 X 12 Hex Head Cap Screw) x 12</td>
			<td>FAS</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Jumper wires (for Arduino Nano)</td>
			<td>ELEGOO</td>
			<td>EL-CP-004</td>
			<td></td>
		</tr>
		<tr>
			<td>LCD screen</td>
			<td>JANSANE</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>M3 Hex Socket Head Cap Screws x 12</td>
			<td>Shenzhen 
			Baishichuangyou 
			Technology co.Ltd</td>
			<td>310luosditaozhuang</td>
			<td></td>
		</tr>
		<tr>
			<td>M3 Stainless SteelMachine screws Flat Head Hex Socket Cap Screws (30 mm) x 36</td>
			<td>Still Awake</td>
			<td>a52400001</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick disconnect terminal connectors</td>
			<td>IEUYO</td>
			<td>22010064</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis Buffer (10x)</td>
			<td>Cell Signaling</td>
			<td>46232</td>
			<td></td>
		</tr>
		<tr>
			<td>Terminal adapter shield Expansion board for Arduino Nano&#160; 12&#34; x 24&#34;</td>
			<td>Shenzhen 
			Weiyapuhua 
			Technology</td>
			<td>60-026-3</td>
			<td></td>
		</tr>
	</tbody>
</table>

mechanical dissociation of tissues for single cell analysis using a motorized device

At kunne isolere og forberede enkeltceller til analyse af vævsprøver er hurtigt blevet afgørende for nye biomedicinske opdagelser og forskning. Manuelle protokoller til enkeltcelleisolering er meget tidskrævende og tilbøjelige til brugervariation. Automatiserede mekaniske protokoller er i stand til at reducere behandlingstid og prøvevariabilitet, men er ikke let tilgængelige eller omkostningseffektive i forskningsindstillinger med lavere ressourcer. Enheden beskrevet her blev designet til halvautomatisk vævsdissociation ved hjælp af kommercielt tilgængelige materialer som et billigt alternativ til akademiske laboratorier. Der er givet instruktioner til fremstilling, samling og betjening af enhedens design. Dissociationsprotokollen producerer pålideligt enkeltcellesuspensioner med sammenlignelige celleudbytter og prøvelevedygtighed til manuelle præparater på tværs af flere musevæv. Protokollen giver mulighed for at behandle op til 12 vævsprøver samtidigt pr. enhed, hvilket gør undersøgelser, der kræver store prøvestørrelser, mere håndterbare. Den ledsagende software giver også mulighed for tilpasning af enhedsprotokollen for at imødekomme forskellige væv og eksperimentelle begrænsninger.

Single-cell analysis has rapidly become crucial for new biomedical discoveries, whether for applications such as flow cytometry, identifying different cell types, single-cell sequencing, or for identifying genomic or transcriptomic variations between cells1. Performing such cell isolations from tissues of interest&#160;requires mincing dissected tissues and pushing them through a fine cell strainer to filter out connective tissue from the desired cells (Figure 1A). Isolating adherent cell types, such as dendritic cells or macrophages, or cells from particularly fibrous tissues, requires additional mechanical or enzymatic separation steps2,3,4. This process is generally done manually, making it highly time-consuming and prone to user variability when assessing cell yields and sample viability. Therefore, it is crucial to introduce customizable options for automated tissue dissociation. While some attempts have been made to design such systems, the existing options are not always readily accessible, particularly in academic labs and lower-resource settings, largely due to the cost-prohibitive nature of these devices5. Furthermore, these devices are not always customizable to the individual needs of a research group6.
Here, a tissue dissociator device was designed to automate the digestion of whole tissues or tissue pieces into single-cell suspensions with the aid of digestive enzymes and mechanical disruption. This device can be easily assembled in the lab, placed into heating or cooling chambers for temperature regulation, customized for the required number of tissues to dissociate, and programmed with the desired dissociation protocols. The broad use of this device could significantly improve the reproducibility of cell extraction protocols and provide a time-saving alternative to manual dissociation.
The design allows for the simultaneous digestion of up to 12 tissues through an automated process. The device is composed of 12 individual motors wired in parallel and powered by a standard wall plug through an AC/DC adapter with an adjustable voltage dial to control the rotation/speed of the motors. The motors turn a hex bolt that fits snugly into the top of the C-tubes. The C-tubes are held in place by downward tension on an acrylic plate that latches on either side to the top plate where the motors are secured (Figure 1B). Because the motors are wired in parallel, their speed at any given voltage should not vary much, but the load (the number of C-tubes mounted on the device) will affect speed even when the voltage is kept constant. To measure rotations per minute (rpm), a tachometer has been incorporated using a hall effect sensor and a fixed magnet on one of the motor shafts (Supplementary Figure 1). The CAD files for building motor arrays are provided in Supplementary Coding File 1. Also included is a programmable switch to reverse the direction of rotation by reversing the positive/negative charges delivered to the motors. All of these features are integrated using coded software (Arduino IDE software, see Table of Materials) on an Arduino Nano (Supplementary Coding File 2). Using connected buttons and an LCD panel (Supplementary Figure 2), it is possible to create and run saved and custom protocols, automatically reverse the rotational direction at specified times of a protocol, adjust speed using the voltage (Supplementary Figure 3), and display the current motor speed and time left to complete a programmed protocol (Supplementary Figure 4).
For the present study, single-cell suspensions were prepared using both mechanical-enzymatic tissue dissociation with this device and manual-enzymatic tissue dissociation to determine differences, if any, in cells recovered for downstream applications. The cell preparations were evaluated based on total cell yields per tissue and&#160;percent cell viability. Flow&#160;cytometry was used to compare potential differences in surface marker expression. Data were analyzed using graphing and statistical analysis software. Unpaired Welch t-tests were used to compare pairs of samples or groups, with sample sizes n &#62; 4 mice representing 2 replicate experiments. Detailed instructions for the fabrication and assembly of this device can be found in Supplementary File 1. Materials needed for this protocol are listed in the Table of Materials.

Forfatter Spotlight: Fremskridt inden for nanopartikelteknologi til lægemiddellevering og immunterapi

Mekanisk dissociation af væv til enkeltcelleanalyse ved hjælp af en motoriseret enhed

Our lab develops nanoparticles as tools to study biological barriers and to overcome these barriers at mucosal surfaces to improve drug delivery and immunotherapy. One of the most notable developments is the COVID-19 vaccines by Pfizer, BioNTech, and Moderna that use lipid nanoparticle formulations to deliver mRNA for vaccination. There are a lot of ongoing efforts to improve immunotherapies like vaccines and cancer treatments using nanoparticles, as these formulations could improve target delivery to immune cells and reduce the systemic side effects of immunotherapies.Single cell analysis is an essential component of immunology in biomedical research, one of the most common techniques being flow cytometry. Performing such cell isolations from tissues of interest requires reliable tissue dissociation. Making laboratory research more accessible without compromising quality and accuracy can be challenging.Automating processes makes technical and labor intensive steps easier to learn and allows trainees to gain experience doing more sophisticated analyses without affecting the reliability of results. The main advantage of this protocol and the accompanying device design is its customizability and cost effectiveness. The ability to process up to 12 tissues simultaneously reduces processing times.This low cost alternative makes this technology more accessible in lower resourced research settings.

Denne halvautomatiske mekaniske protokol kan replikere resultater fra eksperimenter, hvor celler blev behandlet manuelt. Cellesuspensioner fremstillet ved hjælp af denne enhed og ved manuel dissociation viser sammenlignelige celleudbytter og prøvelevedygtighed på tværs af muselunge-, nyre- og hjertevæv (figur 2A, B). Populationer af immunceller, såsom T-celler og dendritiske celler, blev ikke signifikant påvirket af en forskel i isolationsprotokol (<strong class="xfig...

Watch this Scientific Journal Video about Mechanical Dissociation of Tissues for Single Cell Analysis Using a Motorized Device at JoVE.com

Der tilvejebringes en generel protokol for den kombinerede enzymatiske og halvautomatiske mekaniske dissociation af væv til generering af enkeltcellesuspensioner til downstream-analyser, såsom flowcytometri. Instruktioner til fremstilling, montering og drift af den billige mekaniske enhed, der er udviklet til denne protokol, er inkluderet.

Being able to isolate and prepare single cells for the analysis of tissue samples has rapidly become crucial for new biomedical discoveries and research. ...

Vores laboratorium udvikler nanopartikler som værktøjer til at studere biologiske barrierer og overvinde disse barrierer på slimhindeoverflader for at forbedre lægemiddelafgivelse og immunterapi. En af de mest bemærkelsesværdige udviklinger er COVID-19-vaccinerne fra Pfizer, BioNTech og Moderna, der bruger lipidnanopartikelformuleringer til at levere mRNA til vaccination. Der er mange igangværende bestræbelser på at forbedre immunterapier som vacciner og kræftbehandlinger ved hjælp af nanopartikler, da disse formuleringer kan forbedre mållevering til immunceller og reducere de systemiske bivirkninger af immunterapier.Enkeltcelleanalyse er en væsentlig komponent i immunologi inden for biomedicinsk forskning, hvor en af de mest almindelige teknikker er flowcytometri. Udførelse af sådanne celleisoleringer fra væv af interesse kræver pålidelig vævsdissociation. Det kan være en udfordring at gøre laboratorieforskning mere tilgængelig uden at gå på kompromis med kvalitet og nøjagtighed.Automatisering af processer gør tekniske og arbejdskrævende trin lettere at lære og giver praktikanter mulighed for at få erfaring med at udføre mere sofistikerede analyser uden at påvirke pålideligheden af resultaterne. Den største fordel ved denne protokol og det ledsagende enhedsdesign er dens tilpasningsevne og omkostningseffektivitet. Evnen til at behandle op til 12 væv samtidigt reducerer behandlingstiderne.Dette billige alternativ gør denne teknologi mere tilgængelig i forskningsmiljøer med færre ressourcer.

mechanical-dissociation-tissues-for-single-cell-analysis-using

Research

JoVE Journal

Bioengineering

Mechanical Dissociation of Tissues for Single Cell Analysis Using a Motorized Device

Being able to isolate and prepare single cells for the analysis of tissue samples has rapidly become crucial for new biomedical discoveries and research. Manual protocols for single-cell isolations are highly time-consuming and prone to user variability. Automated mechanical protocols are able to reduce processing time and sample variability but aren't easily accessible or cost-effective in lower-resourced research settings. The device described here was designed for semi-automated tissue dissociation using commercially available materials as a low-cost alternative for academic laboratories. Instructions to fabricate, assemble, and operate the device design have been provided. The dissociation protocol reliably produces single-cell suspensions with comparable cell yields and sample viability to manual preparations across multiple mouse tissues. The protocol provides the ability to process up to 12 tissue samples simultaneously per device, making studies requiring large sample sizes more manageable. The accompanying software also allows for customization of the device protocol to accommodate varying tissues and experimental constraints.

A general protocol for the combined enzymatic and semi-automated mechanical dissociation of tissues to generate single-cell suspensions for downstream analyses, such as flow cytometry, is provided. Instructions for the fabrication, assembly, and operation of the low-cost mechanical device developed for this protocol are included.

Semi-Automated Mechanical Dissociation of Mouse Tissues to Generate Single-Cell Suspensions

To begin, place the dissected mouse tissue in a cold cell culture medium containing 5%FBS. Using sharp dissection scissors, chop the tissue until approximately five millimeters fragments are obtained. Transfer the chopped fragments to a C-tube and add one milliliter of cell culture media.Load the C-tube onto the motorized device and fit the tube holder plate over the D-shaped tube bottoms. Latch the tension arms into the acrylic plate to secure the tubes to the motor plate. To set a custom program on the motorized device from the main menu, press the blue button to choose custom mode.In the first custom mode menu, press the green button to specify the duration of forward rotation to 30 seconds. Then press the blue button to select the on screen value. In the second custom mode menu, press the green button to specify the duration of reverse rotation to 10 seconds.Then press the blue button to select the on screen value. In the third custom mode menu, press the red button to specify the selection loops to four times. Then press the blue button to select the on screen value.Adjust the voltage control dial to 200 RPM and start the custom program. Next, add digestion enzyme in four milliliters of cold culture media containing dissected tissues. Again, load the C-tube onto the device and repeat the custom program as demonstrated previously.After the run, transfer the device with the loaded tubes to a 37 degree Celsius incubator for 45 minutes. Next, load the C-tube onto the device and set the custom program at 50 RPM with forward rotation to 270 seconds. Reverse rotation to 30 seconds and looping nine times.Add EDTA to a five millimolar final concentration to the sample. Set the custom program at 100 RPM with forward rotation to 30 seconds. Reverse rotation to 10 seconds and looping twice.Next, pass the cell suspension through a 70 micrometer cell strainer and collect the filtrate in a 50 or 15 milliliter tube. Centrifuge the collected cell suspension at 300 G for five minutes at four degrees Celsius and discard the supernatant. Re-suspend the pellet in one milliliter of red blood cell lysis buffer, and incubate for one minute.Neutralize the lysis buffer with nine milliliters of cold PBS. Again, centrifuge the cells and re-suspend the pellet in the desired buffer or media. Cell suspension prepared with motorized device and manual dissociation exhibited comparable cell viability and yields across mouse lung, kidney, and heart tissues.Immune cell populations like T-cells and dendritic cells were not significantly affected by the isolation protocol. Surface marker expression also showed similar frequencies of isolated T-cells in mean fluorescence intensity for the antigen presentation marker MHC II in dendritic cells between manual and device isolation.