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

Summary

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.

Abstract

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.

Introduction

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 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 steps^2,3,4. 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 percent cell viability. Flow 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 > 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.

Protocol

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.

1. Remove dissected tissue and place it in cold cell culture media with 5% fetal bovine serum (FBS).
2. Chop the tissue using sharp dissection scissors until fragments are smaller than 1 mm in any dimension.
3. Transfer the minced tissue to a 15 mL conical tube and add 5 mL of media.
4. 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 µg/mL).
5. Incubate on a shaker or rocker for 1 h at 37 °C with gentle agitation throughout the incubation. Pipette the sample up and down 100 times.
6. 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.
7. Pass the sample through a 70 µm cell strainer and collect it in a 50 mL or 15 mL tube.
8. Centrifuge the collected cells at 300 x g for 5 min at 4 °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.
9. Neutralize the lysis buffer by adding 9 mL of cold 1X Phosphate Buffered Saline (PBS).
10. Centrifuge the collected cells again at 300 x g for 5 min at 4 °C. Remove the supernatant using a pipette and resuspend cells in the desired buffer or media.

2. Semi-automated mechanical dissociation

1. 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.
2. 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.
3. Load the C-tube onto the device. Fit the tube holder plate (Supplementary Figure 1A) over the D-shaped tube bottoms.
4. Secure the tubes to the motor plate by latching the tension arms into the acrylic plate (Supplementary Figure 3).
5. Set a custom program: Forward, 30 s; Reverse, 10 s; Loop, 4 times.
   1. Choose Custom Mode from the Main Menu by pressing the blue button.
   2. 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.
   3. 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.
   4. 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).
6. 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).
7. Add digestion enzymes in 4 mL 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 µg/mL).
8. 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.
9. Transfer the device with the tubes still loaded to a 37 °C incubator for 45 min.
10. 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.
11. Add EDTA to achieve a 5 mM concentration in the sample.
12. 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.
13. Push the sample through a 70 µm cell strainer and collect it in a 50 mL or 15 mL tube.
14. Centrifuge the collected cells at 300 x g for 5 min at 4 °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.
15. Neutralize the lysis buffer with 9 mL of cold Phosphate Buffered Saline (PBS).
16. Centrifuge the collected cells again at 300 x g for 5 min at 4 °C. Discard the supernatant using a pipette and resuspend cells in the desired buffer or media.

3. Data analysis

1. Analyze the data using a graphing and statistical analysis software (see Table of Materials).
2. Use unpaired Welch t-tests to compare pairs of samples or groups, with sample sizes n > 4 mice representing 2 replicate experiments.
   NOTE: Differences were considered statistically significant when (p < 0.05). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Representative Results

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). Similarly, the analysis of surface marker expression on these immune cell populations shows similar frequencies of isolated T cells (Figure 3A) and comparable mean fluorescence intensity for the antigen presentation marker MHC-II in dendritic cells (Figure 3B) between manual and device isolation.

Figure 1: Process diagrams. (A) Steps in the process of preparing single cell suspension using a simple dissociator device vs. manual dissociation. (B) Dissociator device design and features. Please click here to view a larger version of this figure.

Figure 2: Cell yield and viability between manual and mechanical dissociation protocols. (A) Viability and (B) yield of cells from different tissues immediately after preparation of cell suspension counted by a hemocytometer before flow staining. (C) Cell counts of immune cell populations identified by flow cytometry. Unpaired Welch t-tests were used to compare pairs of samples or groups. Differences were considered statistically significant when p < 0.05. *p< 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars represent SEM. Please click here to view a larger version of this figure.

Figure 3: Comparison of immunophenotypic analysis of single cell suspensions. Flow cytometry comparison of surface marker expression in single cell suspensions prepared using a mechanical dissociator device vs. manual dissociation. (A) Lung CD4+ and CD8+ T Cell populations. (B) Mean fluorescence intensity (MFI) of surface MHC-II on lung dendritic cells. Unpaired Welch t-tests were used to compare pairs of samples or groups. Differences were considered statistically significant when p < 0.05. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars represent SEM. Please click here to view a larger version of this figure.

Supplementary Figure 1: Motor assembly. (A) Images of acrylic sheet CAD files. (B) Assembly of motor/coupler/hex bolt/motor spacer unit onto the motor plate. (C) RPM Sensor. (D) Motor array. (E) Motor wiring diagram. Please click here to download this File.

Supplementary Figure 2: Control panel assembly. (A) Image of Control panel CAD file. (B) Control panel/power supply wiring diagram. Please click here to download this File.

Supplementary Figure 3: Device operation. (A) C-Tubes loaded onto the device and secured with the tube holder plate and tension arms. (B) Variable voltage regulator with dial. Please click here to download this File.

Supplementary Figure 4: Software operation - control panel modes. (A) LCD screen displays and button pushes for various modes of device operation. Please click here to download this File.

Supplementary File 1: Instructions for device fabrication, assembly, and operation. Please click here to download this File.

Supplementary Coding File 1: CAD files for building motor arrays. This file contains CAD files for building motor arrays of 2, 4, 6, 8, 10, and 12. Refer to Supplementary File 1 for details. Please click here to download this File.

Supplementary Coding File 2: Software code for Arduino Nano. Please click here to download this File.

Discussion

This device was designed for easy assembly in the 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 affected by the increased time required for manual dissociation protocols. In research institutions and industry settings, devices like this and other commercially available devices allow for faster and customizable tissue processing; however, commercial devices are over 10 times the cost of the device described here, limiting their accessibility, particularly in low-resource research settings^8,9. In academic laboratories, this device could enable trainees (undergraduate and graduate students) to complete more sophisticated research by reducing the time needed to process tissues for in vivo analyses.

The mechanical dissociation protocol described here was adapted from a protocol regularly used to isolate immune cells from lung tissues⁷. Time-consuming manual steps, such as fine mincing and numerous or vigorous pipetting, were replaced with automated mechanical processing, saving both time and effort required to produce similar results to the manual protocol. This could prove to be ergonomically beneficial for the end user because repetitive pipetting, as prescribed in the manual protocol, could contribute to the development of neuromuscular conditions, such as carpal tunnel syndrome¹⁰.

This semi-automated process can be used to generate cell suspensions for various applications, including flow cytometry and cell culture. It is important to note that all samples should be kept cold or on ice when not incubating at 37 °C, as this could affect cell viability. The enzymes, rotational speeds, and incubation times prescribed in the mechanical protocol may be customized as needed for adaptation to other protocols or desired target cells. However, the minimum and maximum rotational speeds of the device are limited by the specifications of the motors used. For speeds slower than 50 rpm or faster than 200 rpm, motors with a lower or higher maximum speed should be chosen, respectively.

Recommendation for non-engineering laboratories
This device was built in collaboration with Terrapin Works, the fabrication and prototyping center housed in the Engineering department at the University of Maryland. If available, the mechanical, electrical, and software engineers at any academic institution should find this design relatively easy to assemble. Alternatively, a university's machine shop (if available) or any local mechanical, electrical, and/or software engineering laboratories may be able to help with the fabrication or advise on additional available resources.

Future technical improvements
Software code has not been developed to eliminate the manual voltage dial and automatically adjust the motor speed through programming alone. This is possible but would require hardware additions, including a variable voltage regulator, and more sophisticated software to incorporate this feature into the preset and custom program modes.

Materials

Name	Company	Catalog Number	Comments
¼ inch acrylic sheet  12" x 24"	Acrylic Mega Store	N/A
½ inch acrylic sheet 12" x 12"	SimbaLux	SL-AS13-12x12
12 G stainless steel wire (for tension arms)	Everbilt	1000847413
16 G electrical wire (stranded)	Best Connections	N/A
2 x 3 mm magnet	SU-CRO0587	N/A
2-channel relay board (to reverse polarity of current to motors)	AEDIKO	AE06233
37 mm Diameter DC Motors (12 V, 200 rpm) x 12	Greartisan	N/A	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" x 0.55") (D x L) Gearbox Size: 37 x 25 mm (1.46" x 0.98") (D x L) Motor Size: 36.2 x 33.3 mm (1.43" x 1.31") (D x L) Mounting Hole Size: M3 (not included)
AC/C Power Adapter with variable voltage controller   (5 Amps, 3-12 volts)	Mo-gu	J19091-2-MG-US
AC-DC 5 V 1 A Precision buck converter step down transformer	Walfront	1A	(power adapter for powering Arduino Nano)
Arduino Nano   (Lafvin)	LAFVIN	8541582500
Buttons	Awpeye	Push-button
C57BL6/J mice	Jackson Laboratory
Collagenase 4	Worthington	CLS4 LS004188
Collagenase D	Roche	11088866001
DMEM (Dulbecco's Modified Eagle Medium)	Corning	10-013-CV
DNAse	Roche	11284932001
Double sided foam tape	SANKA	N/A
Double Sided prototyping circuit board	deyue	N/A
EDTA	Sigma- Aldrich	E7889
Electrical solder and soldering iron	LDK	1002P
Electrical Tape	3M	03429NA
FBS (Fetal Bovine Serum)	Gibco	16140089
gentleMACS C Tubes	Miltenyi	130-093-237
Graphpad Prism	GraphPad, La Jolla, CA		Graphing and statistical analysis software
Hall effect sensor Dimensions : 0.79 x 0.79x 0.39 inches	SunFounder	43237-2
Hex Coupler 6 mm Bore Motor Brass x 2 x 12	Uxcell	N/A
Hex head bolts (M4-.70 X 12 Hex Head Cap Screw) x 12	FAS	N/A
Jumper wires (for Arduino Nano)	ELEGOO	EL-CP-004
LCD screen	JANSANE	N/A
M3 Hex Socket Head Cap Screws x 12	Shenzhen Baishichuangyou Technology co.Ltd	310luosditaozhuang
M3 Stainless SteelMachine screws Flat Head Hex Socket Cap Screws (30 mm) x 36	Still Awake	a52400001
Quick disconnect terminal connectors	IEUYO	22010064
Red Blood Cell Lysis Buffer (10x)	Cell Signaling	46232
Terminal adapter shield Expansion board for Arduino Nano  12" x 24"	Shenzhen Weiyapuhua Technology	60-026-3