Mar 06, 2026
Generation and collection of MTSs
- Guangli Suo1
- 1CAS Key Laboratory of Nano-Bio Interface, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences
- MTS
Protocol Citation: Guangli Suo 2026. Generation and collection of MTSs. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5q796v1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 06, 2026
Last Modified: March 06, 2026
Protocol Integer ID: 262753
Keywords: collection of mtss, collection of mt, definition of mfr, mtss, mt, mfr, collection
Abstract
The definition of MFR and the collection of MTS。
Guidelines
The generation of MTSs in this study utilized a panel of seven BRC cell lines, namely MDA-MB-231 (M231), MCF7, MDA-MB-435S, BT-549, AU-565, MDA-MB-453 and MDA-MB-435. Following trypsinization of the cells from monolayer culture, they were collected and seeded in the MWC-chip for subsequent culture in the Sel-Medium for 3 days, followed by an additional two-day culture under SG-Medium conditions to induce MTSs formation.
After 5 days of culture, the SG-Medium was aspirated and all cells, including MTSs and single live cells, as well as cell debris, were collected as a mixture from the microwells by washing with PBS. The MTSs ranging in diameter from 30 to 70 μm were sorted using hydrodynamics actuated cell sorting (HACS) technology, or individual MTS and other residual cells (RCs) within a microwell were accurately picked separately using CytoSTAR (Livingchip) for subsequent experiments.
Materials
- Ice-cold phosphate-buffered saline (PBS) supplemented with penicillin-streptomycin (100 U/mL)
- Collagenase I, II, IV
- DNase (Thermo Fisher Scientific)
- RBC lysis buffer (Invivogen)
- Selective medium (Sel-Medium)
- DMEM/F12 selective medium containing 15 mM HEPES buffer (STEMCELL Technologies)
- SG-Medium
- LumaScope 620 (Etaluma)
- BRC cell lines: MDA-MB-231 (M231), MCF7, MDA-MB-435S, BT-549, AU-565, MDA-MB-453, MDA-MB-435
- CytoSTAR (Livingchip)
Troubleshooting
Generation and collection of MTSs
For generation of MTSs derived from clinical BRC tissues, the surgical or biopsy specimens were immediately immersed in ice-cold phosphate-buffered saline (PBS) supplemented with penicillin-streptomycin (100 U/mL) and transported to the laboratory in 24 hours. Necrotic regions and adipose tissue were excised as possible, followed by washing with PBS. The excised tissues were then minced into small pieces and subjected to enzymatic digestion using collagenase I, II, IV along with DNase (Thermo Fisher Scientific) at 37 °C for 1-1.5 hours. In case of any visible red color appeared during sample preparation, erythrolysis was performed with RBC lysis buffer (Invivogen) before the washing step. Typically, 0.5 mL of lysate could digest one piece of biopsy samples with a total weight of approximately 50 mg. The digestion mixture was pipetted every 15 minutes to facilitate cell release. The dissociated cells were collected by passing through a 40 μm filter and then centrifuged at 1,200 rpm for 5 minutes at 4 °C. After centrifugation, the cells were resuspended in selective medium (Sel-Medium), the DMEM/F12 selective medium containing 15 mM HEPES buffer (STEMCELL Technologies). The resuspended cells were then seeded onto one MWC-chip. Following 2 days of culture in Sel-Medium, the medium was replaced with SG-Medium for further culturing of cells for an additional 2-3 days in an incubator at 37 °C, with 5% CO₂. Subsequently, MTSs larger than 30 μm diameter formed within microwells amidst numerous single cells or cell debris. The capacity of MTSs formation on the cell-chip was quantified using the following equation: MTSs formation rate on microwell cell-chip (MFR) = (Number of MTSs on a MWC-chip / Total number of wells within a MWC-chip) × 100%. The success rate of MTS generation from clinical samples used in this study was determined as follows: (Number of samples with MFR 3e 5% / Total number of samples used in this study) × 100%. To capture the growth dynamics of MTSs, cells cultured in SGM were imaged every 5 minutes using LumaScope 620 (Etaluma) over a total duration of 96 hours. The resulting videos were created and analyzed using LumaScope 620 (Etaluma).
The generation of MTSs in this study utilized a panel of seven BRC cell lines, namely MDA-MB-231 (M231), MCF7, MDA-MB-435S, BT-549, AU-565, MDA-MB-453 and MDA-MB-435. Following trypsinization of the cells from monolayer culture, they were collected and seeded in the MWC-chip for subsequent culture in the Sel-Medium for 3 days, followed by an additional two-day culture under SG-Medium conditions to induce MTSs formation.
After 5 days of culture, the SG-Medium was aspirated and all cells, including MTSs and single live cells, as well as cell debris, were collected as a mixture from the microwells by washing with PBS. The MTSs ranging in diameter from 30 to 70 μm were sorted using hydrodynamics actuated cell sorting (HACS) technology, or individual MTS and other residual cells (RCs) within a microwell were accurately picked separately using CytoSTAR (Livingchip) for subsequent experiments.
Protocol references
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