Name: mechanical dissociation of tissues for single cell analysis using a motorized device
Uploaded: 2023-11-10T12:50:00.000+00:00
Duration: 5 min 6 s
Description: Watch this Scientific Journal Video about Mechanical Dissociation of Tissues for Single Cell Analysis Using a Motorized Device at JoVE.com

Funding was received from the Fischell Department of Bioengineering (KM), T32 GM080201 (MA), Vogel Endowed Summer Fellowship (MA), LAM Foundation (KM), and American Lung Association (KM).&#160;The authors wish to thank Michele Kaluzienski for help with editing.

This protocol was approved by the UMD Institutional Animal Care and Use Committee (IACUC). Tissues from 7 to 9-week-old female C57BL/6J mice were used for these studies. The animals were obtained from a commercial source (see Table of Materials).
1. Manual dissociation
NOTE: This step is adapted from Maisel K. et al.7.
<ol>
	<li>Remove dissected tissue and place it in cold cell culture media with 5% fetal bovine serum (FBS).</li>
	<li>Chop the tissue using sharp dissection scissors until fragments are smaller than 1 mm in any dimension.</li>
	<li>Transfer the minced tissue to a 15 mL conical tube and add 5 mL of media.</li>
	<li>Add digestion enzymes based on the tissue and cell types being isolated. For the presented experiments, use Collagenase 4 (1 mg/mL), Collagenase D (1 mg/mL), and DNase (40 &#181;g/mL).</li>
	<li>Incubate on a shaker or rocker for 1 h at 37 &#176;C with gentle agitation throughout the incubation. Pipette the sample up and down 100 times.</li>
	<li>Add EDTA to achieve a sample volume with a 5 mM concentration. Pipette the sample up and down 100 times, and then vigorously pipette the sample up and down 100 times.</li>
	<li>Pass the sample through a 70 &#181;m cell strainer and collect it in a 50 mL or 15 mL tube.</li>
	<li>Centrifuge the collected cells at 300 x g for 5 min at 4 &#176;C. Remove the supernatant using a pipette and resuspend cells in 1 mL of red blood cell lysis buffer. Incubate for 1 min at room temperature.</li>
	<li>Neutralize the lysis buffer by adding 9 mL of cold 1X Phosphate Buffered Saline (PBS).</li>
	<li>Centrifuge the collected cells again at 300 x g for 5 min at 4 &#176;C. Remove the supernatant using a pipette and resuspend cells in the desired buffer or media.</li>
</ol>
2. Semi-automated mechanical dissociation
<ol>
	<li>Place dissected tissue in cold cell culture media with 5% fetal bovine serum (FBS). 
	NOTE: This protocol is intended for cell isolation from solid tissues.</li>
	<li>Chop the tissue using sharp dissection scissors until tissue fragments are approximately 5 mm in any dimension. Transfer the chopped tissue to a C-tube and add 1 mL of media.</li>
	<li>Load the C-tube onto the device. Fit the tube holder plate (Supplementary Figure 1A) over the D-shaped tube bottoms.</li>
	<li>Secure the tubes to the motor plate by latching the tension arms into the acrylic plate (Supplementary Figure 3).</li>
	<li>Set a custom program: Forward, 30 s; Reverse, 10 s; Loop, 4 times.
	<ol>
		<li>Choose Custom Mode from the Main Menu by pressing the blue button.</li>
		<li>In the first custom mode menu, specify the duration of forward rotation to 30 s by pressing the red button until the value on the screen reads 30 s. Press the blue button to select this value and advance to the next menu screen.</li>
		<li>In the second custom mode menu, specify the duration of reverse rotation to 10 s by pressing the red button until the value on the screen reads 10 s. Press the blue button to select this value and advance to the next menu screen.</li>
		<li>In the third custom mode menu, specify the number of times to repeat the selections set in steps 2.5.2-2.5.3 by pressing the red button until the value on the screen reads 4X. Press the blue button to select this value and start the program. 
		NOTE: Custom programs are not stored in the device memory but can be programmed as one of the preset programs for faster operation (device programming instructions are provided in Supplementary File 1).</li>
	</ol>
	</li>
	<li>Run the custom program at 200 rpm by adjusting the voltage control dial (Supplementary Figure 3) until the calculated rpm value in the bottom right corner of the LCD Screen reads 200 (Supplementary Figure 4).</li>
	<li>Add digestion enzymes in 4 mL&#160;media according to the tissue and cell types being isolated. 
	NOTE: For the presented experiments, use Collagenase 4 (1 mg/mL), Collagenase D (1 mg/mL), and DNase (40 &#181;g/mL).</li>
	<li>Load the C-tube onto the device and repeat steps 2.3-2.6 to run the following program at 200 rpm: Forward, 30 s; Reverse, 10 s; Loop, 4 times.</li>
	<li>Transfer the device with the tubes still loaded to a 37 &#176;C incubator for 45 min.</li>
	<li>Load the C-tube onto the device and repeat steps 2.3-2.6 to run the following program at 50 rpm: Forward, 270 s; Reverse, 30 s; Loop, 9 times.</li>
	<li>Add EDTA to achieve a 5 mM concentration in the sample.</li>
	<li>Load the C-tube onto the device and repeat steps 2.3-2.6 to run the following program at 100 rpm: Forward, 30 s; Reverse, 10 s; Loop, 2 times.</li>
	<li>Push the sample through a 70 &#181;m cell strainer and collect it in a 50 mL or 15 mL tube.</li>
	<li>Centrifuge the collected cells at 300 x g for 5 min at 4 &#176;C. Discard the supernatant using a pipette and resuspend cells in 1 mL of red blood cell lysis buffer. Incubate for 1 min at room temperature.</li>
	<li>Neutralize the lysis buffer with 9 mL of cold Phosphate Buffered Saline (PBS).</li>
	<li>Centrifuge the collected cells again at 300 x g for 5 min at 4 &#176;C. Discard the supernatant using a pipette and resuspend cells in the desired buffer or media.</li>
</ol>
3. Data analysis
<ol>
	<li>Analyze the data using a graphing and statistical analysis software (see Table of Materials).</li>
	<li>Use unpaired Welch t-tests to compare pairs of samples or groups, with sample sizes n &#62; 4 mice representing 2 replicate experiments. 
	NOTE: Differences were considered statistically significant when (p &#60; 0.05). *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001, ****p &#60; 0.0001.</li>
</ol>

Semi-Automated Mechanical-Enzymatic Tissue Dissociation for Single-Cell Analysis

<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>

The authors have no conflicts of interest to disclose.

This device was designed for easy assembly in the&#160;research setting to provide single-cell suspensions from whole tissues for subsequent single-cell analysis. The features, while basic, are sufficient to meet the needs of researchers in academic settings and beyond. A key benefit of using this device is its potential to improve the preparation of single-cell suspensions by reducing variability. Additionally, the ability to process 12+ samples simultaneously could enhance sample-to-sample consistency, which may be aff...

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.

<table><tbody><tr><td>&amp;frac14; inch acrylic sheet&amp;nbsp; 12&quot; x 24&quot;</td><td>Acrylic Mega Store</td><td>N/A</td><td></td></tr><tr><td>&amp;frac12; inch acrylic sheet 12&quot; x 12&quot;</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&amp;nbsp;&amp;nbsp;</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&lt;br/&gt; Reduction Ratio: 1:22&lt;br/&gt; Rated Current: 0.1 A&lt;br/&gt; D Shaped Output Shaft Size: 6 x 14mm (0.24&quot; x 0.55&quot;) (D x L)&lt;br/&gt; Gearbox Size: 37 x 25 mm (1.46&quot; x 0.98&quot;) (D x L)&lt;br/&gt; Motor Size: 36.2 x 33.3 mm (1.43&quot; x 1.31&quot;) (D x L)&lt;br/&gt; Mounting Hole Size: M3 (not included)</td></tr><tr><td>AC/C Power Adapter with variable voltage controller&amp;nbsp;&amp;nbsp; (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&amp;nbsp;&amp;nbsp; (Lafvin)</td><td>LAFVIN</td><td>8541582500</td><td></td></tr><tr><td>Buttons&amp;nbsp;</td><td>Awpeye</td><td>Push-button</td><td></td></tr><tr><td>C57BL6/J mice&amp;nbsp;</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&#039;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&lt;br/&gt; Baishichuangyou&lt;br/&gt; 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&amp;nbsp; 12&quot; x 24&quot;</td><td>Shenzhen&lt;br/&gt; Weiyapuhua&lt;br/&gt; Technology</td><td>60-026-3</td><td></td></tr></tbody></table>

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.

This semi-automated mechanical protocol can replicate results from experiments in which cells were processed manually. Cell suspensions prepared using this device and by manual dissociation show comparable cell yields and sample viability across mouse lung, kidney, and heart tissues (Figure 2A,B). Populations of immune cells, such as T cells and dendritic cells, were not significantly affected by a difference in isolation protocol (Figure 2C). S...

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

Author Spotlight: Advancements in Nanoparticle Technology for Drug Delivery and Immunotherapy

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.

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. ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

880 Views.  University of Maryland. 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.

Video: Author Spotlight: Advancements in Nanoparticle Technology for Drug Delivery and Immunotherapy

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual dissociations the gentleMACS Dissociator allows one to dissociate tissue very efficiently under controlled and reproducible conditions.  The gentleMACS Dissociator can optimally dissociate mouse spleen, combining timesaving and standardization with user-safety.

This video describes how to dissociate mouse spleens using the gentleMACS Dissociator, an automated bench-top device that can mechanically disrupt tissues using special tubes to produce viable cell suspensions. Following dissociation, spleens are filtered, centrifuged, and resuspended for further applications.

This video describes a simple, time-saving technique for automated tissue dissociation using the gentleMACS Dissociator to prepare single-cell suspensions of mouse splenocytes.

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

Biology

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Developmental Biology

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Pancreatic Tissue Dissection to Isolate Viable Single Cells

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for approximately 90% of the pancreas and includes cells that produce and secrete digestive enzymes. The digestive enzymes are produced in the pancreatic acinar cells, stored in vesicles called zymogens, and are then released into the duodenum via the pancreatic duct to initiate metabolic processes. The enzymes produced by the acinar cells can kill cells or degrade cell-free RNA. In addition, acinar cells are fragile, and common dissociation protocols result in a large number of dead cells and cell-free proteases and RNases. Therefore, one of the biggest challenges in pancreatic tissue digestion is recovering intact and viable cells, especially acinar cells. The protocol presented in this article shows a two-step method that we developed to meet this need. The protocol can be used to digest normal pancreata, pancreata that include pre-malignant lesions, or pancreatic tumors that include a large number of stromal and immune cells.

Pancreatic metaplastic cells are precursors of malignant cells that give rise to pancreatic tumors. However, isolating intact viable pancreatic cells is challenging. Here, we present an efficient method for pancreatic tissue dissociation. The cells can then be used for single-cell RNA sequencing (scRNA-seq) or for two- or three-dimensional co-culturing.

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for ...

Cancer Research

Tumor Dissociation Protocol for Single-Cell RNA Sequencing Analysis

Dissociation of Human and Mouse Tumor Tissue Samples for Single-cell RNA Sequencing

Human tumor samples hold a plethora of information about their microenvironment and immune repertoire. Effective dissociation of human tissue samples into viable cell suspensions is a required input for the single-cell RNA sequencing (scRNAseq) pipeline. Unlike bulk RNA sequencing approaches, scRNAseq enables us to infer the transcriptional heterogeneity in tumor specimens at the single-cell level. Incorporating this approach in recent years has led to many discoveries, such as identifying immune and tumor cellular states and programs associated with clinical responses to immunotherapies and other types of treatments. Moreover, single-cell technologies applied to dissociated tissues can be used to identify accessible chromatin regions T and B cell receptor repertoire, and the expression of proteins, using DNA barcoded antibodies (CITEseq).
The viability and quality of the dissociated sample are critical variables when using these technologies, as these can dramatically affect the cross-contamination of single cells with ambient RNA, the quality of the data, and interpretation. Moreover, long dissociation protocols can lead to the elimination of sensitive cell populations and the upregulation of a stress response gene signature. To overcome these limitations, we devised a rapid universal dissociation protocol, which has been validated on multiple types of human and murine tumors. The process begins with mechanical and enzymatic dissociation, followed by filtration, red blood lysis, and live dead enrichment, suitable for samples with a low input of cells (e.g., needle core biopsies). This protocol ensures a clean and viable single-cell suspension paramount to the successful generation of Gel Bead-In Emulsions (GEMs), barcoding, and sequencing.

Author Spotlight: Exploring Strategies for Successful Immune Response Against Tumors

This protocol outlines the procedure for rapidly dissociating human and mouse tumor samples for single-cell RNA sequencing.

Human tumor samples hold a plethora of information about their microenvironment and immune repertoire. Effective dissociation of human tissue samples into ...

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in preparation for detailed downstream analysis such as flow cytometry or single-cell sequencing. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination thereof. The delicate nature of neuronal cells can complicate efforts to obtain the highly viable, true single-cell suspension with minimal cellular debris that is required for single-cell analysis. The data demonstrate that this combination of automated mechanical dissociation and enzymatic digestion consistently yields a highly viable (&gt;90%) single-cell suspension, overcoming the aforementioned difficulties. While a few of the steps require manual dexterity, these steps lessen sample handling and potential cell loss. This manuscript details each step of the process to equip other laboratories to successfully dissociate small quantities of neural tissue in preparation for downstream analysis.

This neural cell dissociation protocol is intended for samples with a low amount of starting material and yields a highly viable single-cell suspension for downstream analysis, with optional fixation and staining steps.

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in ...

Neuroscience

A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury

We describe the implementation of spinal cord injury in mice to elicit detrusor-sphincter dyssynergia, a functional bladder outlet obstruction, and subsequent bladder wall remodeling. To facilitate assessment of the cellular composition of the bladder wall in non-injured control and spinal cord injured mice, we developed an optimized dissociation protocol that supports high cell viability and enables the detection of discrete subpopulations by flow cytometry.
Spinal cord injury is created by complete transection of the thoracic spinal cord. At the time of tissue harvest, the animal is perfused with phosphate-buffered saline under deep anesthesia and bladders are harvested into Tyrode&#8217;s buffer. Tissues are minced prior to incubation in digestion buffer that has been optimized based on the collagen content of mouse bladder as determined by interrogation of publicly available gene expression databases. Following generation of a single cell suspension, material is analyzed by flow cytometry for assessment of cell viability, cell number and specific subpopulations. We demonstrate that the method yields cell populations with greater than 90% viability, and robust representation of cells of mesenchymal and epithelial origin. This method will enable accurate downstream analysis of discrete cell types in mouse bladder and potentially other organs.

The goal of this protocol is to apply an optimized tissue dissociation protocol to a mouse model of spinal cord injury and validate the approach for single cell analysis by flow cytometry.

We describe the implementation of spinal cord injury in mice to elicit detrusor-sphincter dyssynergia, a functional bladder outlet obstruction, and ...

Optimized Protocol for Mouse Wound Healing Model with Stem Cell-Derived Exosomes

Mouse Wound Models and Preparation of Single-Cell Suspensions 

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving diverse cell populations and the intricate wound microenvironment, complicates the assessment of treatment effects and their interactions using traditional experimental approaches. Single-cell transcriptome sequencing technology has revolutionized the understanding of cellular functions and mechanisms during wound healing. However, the variability in methodologies for creating mouse wound models introduces confounding factors that can impact experimental results. Furthermore, the process of dissociating mouse skin tissues presents significant technical hurdles, highlighting the critical need for high-quality single-cell suspensions for accurate transcriptome sequencing. Therefore, this study aims to optimize the construction of mouse wound models to accurately assess changes in wound closure while eliminating variables that could influence the subsequent sequencing result. Additionally, the preparation of single-cell suspensions was performed using both enzyme preparation protocols and test kit protocols to optimize cost and effectiveness.

Mouse Wound Models and Preparation of Single-Cell Suspensions

This study presents two crucial experimental techniques: the construction of a murine wound model for assessing wound closure and the preparation of single-cell suspensions from murine skin to preserve cellular viability.

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving ...

Medicine

Mechanical Dissociation: A Method to Obtain Viable Cells from a Tissue

Source: Garaud et. al., <a href="https://jove.unicartagenaproxy.elogim.com/v/52392/a-simple-rapid-protocol-to-non-enzymatically-dissociate-fresh-human">A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes.</a> J. Vis. Exp. (2014).
This video describes the non-enzymatic dissociation of fresh human tissue to obtain viable cells. The technique is used for qualitative and quantitative analysis of CD45 positive cells (lymphocytes/leukocytes) present in various normal and malignant human tissues.

Dysocjacja mechaniczna: metoda uzyskiwania żywotnych komórek z tkanki

Source: Garaud et. al., A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes. J. ...

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

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Pancreatic Tissue Dissection to Isolate Viable Single Cells

Dissociation of Human and Mouse Tumor Tissue Samples for Single-cell RNA Sequencing

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury

Mouse Wound Models and Preparation of Single-Cell Suspensions

Dysocjacja mechaniczna: metoda uzyskiwania żywotnych komórek z tkanki