Name: Forfatter Spotlight: Fremskritt innen nanopartikkelteknologi for medikamentlevering 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 ble mottatt fra Fischell Department of Bioengineering (KM), T32 GM080201 (MA), Vogel Endowed Summer Fellowship (MA), LAM Foundation (KM) og American Lung Association (KM). Forfatterne takker Michele Kaluzienski for hjelp med redigering.

Denne protokollen ble godkjent av UMD Institutional Animal Care and Use Committee (IACUC). Vev fra 7 til 9 uker gamle kvinnelige C57BL/6J-mus ble brukt til disse studiene. Dyrene ble hentet fra en kommersiell kilde (se Materialfortegnelse).
1. Manuell dissosiasjon
MERK: Dette trinnet er tilpasset fra Maisel K. et al.7.
<ol><li>Fjern dissekert vev og legg det i kaldt cellekulturmedium med 5% føtalt bovint s...

Halvautomatisk mekanisk-enzymatisk vevsdissosiasjon for encelleanalyse

<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 enheten ble designet for enkel montering i forskningsinnstillingen for å gi enkeltcellesuspensjoner fra hele vev for påfølgende enkeltcelleanalyse. Funksjonene, selv om de er grunnleggende, er tilstrekkelige til å møte behovene til forskere i akademiske omgivelser og utover. En viktig fordel ved å bruke denne enheten er dens potensial til å forbedre fremstillingen av encellede suspensjoner ved å redusere variabiliteten. I tillegg kan muligheten til å behandle 12+ prøver samtidig forbedre prøve-til-prøve...

Enkeltcelleanalyse har raskt blitt avgjørende for nye biomedisinske oppdagelser, enten for applikasjoner som flowcytometri, identifisering av forskjellige celletyper, enkeltcellesekvensering eller for å identifisere genomiske eller transkriptomiske variasjoner mellom celler1. Å utføre slike celleisoleringer fra vev av interesse krever hakking av dissekerte vev og skyve dem gjennom en fin cellesil for å filtrere ut bindevev fra de ønskede cellene (figur 1A). Isolering av adherente celletyper, som dendrittiske celler eller makrofager, eller celler fra spesielt fibrøst vev, krever ytterligere mekaniske eller enzymatiske separasjonstrinn 2,3,4. Denne prosessen gjøres vanligvis manuelt, noe som gjør den svært tidkrevende og utsatt for brukervariabilitet når man vurderer celleutbytter og prøvens levedyktighet. Derfor er det avgjørende å introdusere tilpassbare alternativer for automatisert vevsdissosiasjon. Mens noen forsøk har blitt gjort for å designe slike systemer, er de eksisterende alternativene ikke alltid lett tilgjengelige, spesielt i akademiske laboratorier og lavere ressursinnstillinger, hovedsakelig på grunn av den kostnadsuoverkommelige naturen til disse enhetene5. Videre kan disse enhetene ikke alltid tilpasses de individuelle behovene til en forskningsgruppe6.
Her ble en vevsdissosiatorenhet designet for å automatisere fordøyelsen av hele vev eller vevstykker til encellede suspensjoner ved hjelp av fordøyelsesenzymer og mekanisk forstyrrelse. Denne enheten kan enkelt monteres i laboratoriet, plasseres i varme- eller kjølekamre for temperaturregulering, tilpasset det nødvendige antall vev for å dissociere, og programmeres med de ønskede dissosiasjonsprotokollene. Den brede bruken av denne enheten kan forbedre reproduserbarheten av celleekstraksjonsprotokoller betydelig og gi et tidsbesparende alternativ til manuell dissosiasjon.
Designet muliggjør samtidig fordøyelse av opptil 12 vev gjennom en automatisert prosess. Enheten består av 12 individuelle motorer koblet parallelt og drevet av en standard veggplugg gjennom en AC / DC-adapter med et justerbart spenningshjul for å kontrollere motorens rotasjon / hastighet. Motorene snur en sekskantbolt som passer godt inn i toppen av C-rørene. C-rørene holdes på plass av nedadgående spenning på en akrylplate som låses på hver side til topplaten der motorene er festet (figur 1B). Fordi motorene er koblet parallelt, bør hastigheten ved en gitt spenning ikke variere mye, men belastningen (antall C-rør montert på enheten) vil påvirke hastigheten selv når spenningen holdes konstant. For å måle rotasjoner per minutt (rpm) er det integrert en turteller ved hjelp av en halleffektsensor og en fast magnet på en av motorakslene (tilleggsfigur 1). CAD-filene for bygging av motormatriser finnes i tilleggskodefil 1. Inkludert er også en programmerbar bryter for å reversere rotasjonsretningen ved å reversere de positive / negative ladningene som leveres til motorene. Alle disse funksjonene er integrert ved hjelp av kodet programvare (Arduino IDE-programvare, se Materialfortegnelse) på en Arduino Nano (Supplementary Coding File 2). Ved hjelp av tilkoblede knapper og et LCD-panel (tilleggsfigur 2) er det mulig å opprette og kjøre lagrede og egendefinerte protokoller, automatisk reversere rotasjonsretningen på bestemte tidspunkter i en protokoll, justere hastigheten ved hjelp av spenningen (tilleggsfigur 3) og vise gjeldende motorhastighet og tid som gjenstår for å fullføre en programmert protokoll (tilleggsfigur 4).
For denne studien ble enkeltcellesuspensjoner fremstilt ved bruk av både mekanisk-enzymatisk vevsdissosiasjon med denne enheten og manuell-enzymatisk vevsdissosiasjon for å bestemme forskjeller, om noen, i celler gjenvunnet for nedstrøms applikasjoner. Cellepreparatene ble evaluert basert på totalt celleutbytte per vev og prosent cellelevedyktighet. Flowcytometri ble brukt for å sammenligne potensielle forskjeller i overflatemarkøruttrykk. Data ble analysert ved hjelp av grafisk og statistisk analyseprogramvare. Uparede Welch t-tester ble brukt til å sammenligne par av prøver eller grupper, med utvalgsstørrelser n > 4 mus som representerer 2 replikere eksperimenter. Detaljerte instruksjoner for fabrikasjon og montering av denne enheten finnes i tilleggsfil 1. Materialer som trengs for denne protokollen er oppført i materialfortegnelsen.

<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

Å kunne isolere og forberede enkeltceller for analyse av vevsprøver har raskt blitt avgjørende for nye biomedisinske funn og forskning. Manuelle protokoller for enkeltcelleisolasjoner er svært tidkrevende og utsatt for brukervariasjon. Automatiserte mekaniske protokoller er i stand til å redusere behandlingstid og utvalgsvariabilitet, men er ikke lett tilgjengelige eller kostnadseffektive i forskningsinnstillinger med lavere ressurser. Enheten beskrevet her ble designet for semi-automatisert vevsdissosiasjon ved bruk av kommersielt tilgjengelige materialer som et billig alternativ for akademiske laboratorier. Instruksjoner for å fremstille, montere og betjene enhetens design er gitt. Dissosiasjonsprotokollen produserer pålitelig enkeltcellesuspensjoner med sammenlignbare celleutbytter og prøvens levedyktighet til manuelle preparater over flere musevev. Protokollen gir muligheten til å behandle opptil 12 vevsprøver samtidig per enhet, noe som gjør studier som krever store prøvestørrelser mer håndterbare. Den medfølgende programvaren tillater også tilpasning av enhetsprotokollen for å imøtekomme varierende vev og eksperimentelle begrensninger.

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: Fremskritt innen nanopartikkelteknologi for medikamentlevering og immunterapi

Mekanisk dissosiasjon av vev for enkeltcelleanalyse ved hjelp av en motorisert enhet

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 halvautomatiserte mekaniske protokollen kan replikere resultater fra eksperimenter der celler ble behandlet manuelt. Cellesuspensjoner fremstilt ved hjelp av denne enheten og ved manuell dissosiasjon viser sammenlignbare celleutbytter og prøvens levedyktighet over musens lunge-, nyre- og hjertevev (figur 2A, B). Populasjoner av immunceller, som T-celler og dendrittiske celler, ble ikke signifikant påvirket av en forskjell i isolasjonsprotokoll (f...

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

En generell protokoll for kombinert enzymatisk og semi-automatisert mekanisk dissosiasjon av vev for å generere encellede suspensjoner for nedstrømsanalyser, slik som flowcytometri, er gitt. Instruksjoner for fabrikasjon, montering og drift av den rimelige mekaniske enheten utviklet for denne protokollen er inkludert.

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

Vårt laboratorium utvikler nanopartikler som verktøy for å studere biologiske barrierer og for å overvinne disse barrierene på slimhinneoverflater for å forbedre legemiddellevering og immunterapi. En av de mest bemerkelsesverdige utviklingene er COVID-19-vaksinene fra Pfizer, BioNTech og Moderna som bruker lipidnanopartikkelformuleringer for å levere mRNA til vaksinering. Det er mange pågående anstrengelser for å forbedre immunterapier som vaksiner og kreftbehandlinger ved hjelp av nanopartikler, da disse formuleringene kan forbedre målleveransen til immunceller og redusere de systemiske bivirkningene av immunterapier.Enkeltcelleanalyse er en viktig komponent i immunologi i biomedisinsk forskning, en av de vanligste teknikkene er flowcytometri. Å utføre slike celleisolasjoner fra vev av interesse krever pålitelig vevsdissosiasjon. Det kan være utfordrende å gjøre laboratorieforskning mer tilgjengelig uten at det går på bekostning av kvalitet og nøyaktighet.Automatisering av prosesser gjør tekniske og arbeidskrevende trinn enklere å lære, og lar lærlinger få erfaring med å gjøre mer sofistikerte analyser uten å påvirke påliteligheten til resultatene. Den største fordelen med denne protokollen og den medfølgende enhetsdesignen er dens tilpassbarhet og kostnadseffektivitet. Evnen til å behandle opptil 12 vev samtidig reduserer behandlingstiden.Dette lavprisalternativet gjør denne teknologien mer tilgjengelig i forskningsmiljøer med lavere ressurser.

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.