Application protocol

Isolation of human tumor cells from tumor tissue samples

In this application protocol, we describe how to isolate untouched human tumor cells in an easy workflow that improves sensitivity and reduces bias in downstream applications, such as next-generation sequencing and tumor cell culture. Solid tumors are dissociated into viable single-cell suspensions and untouched tumor cells are then isolated using MACS® technology.

Materials

The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

For tumor tissue dissociation

  • Tumor Dissociation Kit, human (# 130-095-929)
  • gentleMACS™ Octo Dissociator with Heaters (# 130-096-427)
  • gentleMACS C Tubes (# 130-093-237)
  • RPMI 1640 (# 130-091-440) or DMEM (# 130-091-437) culture media
  • MACS® SmartStrainers (70 µm) (# 130-098-462)
  • MACSmix™ Tube Rotator (# 130‑090‑753) in combination with an incubator at 37 °C
  • (Optional) Red Blood Cell Lysis Solution (10×) (# 130-094-183)
  • (Optional) MACS Tissue Storage Solution (# 130-100-008)
  • (Optional) ART® 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes

For isolation of tumor cells

  • Tumor Cell Isolation Kit, human (# 130-108-339)
  • QuadroMACS™ Starting Kit (LS) (# 130-091-051)
  • Pre-Separation Filters (70 µm) (# 130-095-823)
  • PB Buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2, and 0.5% bovine serum albumin (BSA) by diluting MACS BSA Stock Solution (# 130‑091-376) 1:20 with PBS. Keep buffer cold (2−8 °C). Degas buffer before use, as air bubbles could block the column. Always use freshly prepared buffer. Do not use autoMACS® Running Buffer or MACSQuant® Running Buffer as they contain a small amount of sodium azide that could affect the results.
  • (Optional) Propidium Iodide Solution (# 130-093-233) or 7-AAD for flow cytometry exclusion of dead cells
  • (Optional) Fluorochrome-conjugated antibodies for flow cytometry analysis, e.g., CD326 (EpCAM)-VioBlue®. and CD31-PE. Learn more about our antibodies and dyes.
  • (Optional) Labeling Check Reagent conjugated to, e.g., APC (# 130-095-237) to evaluate purity of sorted cells
  • PBS buffer

Automated protocol

The materials and methods described in this Application are for the manual protocol. Automated cell isolation can be performed with the autoMACS® Pro or the multiMACS™ Cell24 instruments.
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Protocol


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The following instructions are for manual cell enrichment.

Create a viable single-cell suspension from a solid human tumor using the gentleMACS™ Octo Dissociator with Heaters in combination with the Tumor Dissociation Kit, human. Follow the protocol from the kit data sheet.

Download data sheet

Tumor Dissociation Kit, human

Isolate untouched human tumor cells using the Tumor Cell Isolation Kit, human and LS Columns. Follow the protocol of the kit data sheet.

Download the data sheet

Tumor Cell Isolation Kit, human


We recommend filtering the magnetically labeled cell suspension to guarantee it is single-celled before separating it on the column.

  1. Place a Pre-Separation Filter (70 µm) on the LS Column.
  2. Rinse the column 3 times with PBS, ensuring that the filter is pre-wetted.
  3. Apply the cell suspension and PB buffer to the filter on the column.
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General workflow for the rapid isolation of untouched tumor cells. The procedure is based on the comprehensive depletion of cells of non-tumor origin by combining automated tissue dissociation and magnetic cell isolation. A negative selection strategy enables the isolation of the tumor cell population without specific knowledge of surface marker expression on these cells.


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Flow cytometry analysis of isolated human tumor cells.  A comparison of bulk tumor tissue and isolated tumor cells shows elimination of over 95% of the contaminating cells with this protocol, which takes less than 20 minutes.

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Cultures of isolated tumor cells were nearly pure after seven days. Upon magnetic separation, the original bulk (left) and isolated tumor cell (right) fractions were cultured for three to seven days, fixed, and stained. Cells were stained for the human-specific epithelial tumor marker CD326 (EpCAM) and vimentin (human tumor cells were negative for vimentin) to unambiguously identify fibroblasts. Even after seven days, the cultures of isolated tumor cells were nearly pure.

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Accurate detection of loss of heterozygosity events in isolated tumor cells. DNA from bulk tumor or isolated tumor cells was used to produce exome-captured sequencing libraries applying the Nextera® Rapid Capture Exome Kit (Illumina®). For sequencing on a MiSeq® instrument (Illumina) the MiSeq Reagent Kit v3 (150 cycle, Illumina) was utilized to generate 75-bp paired-end reads. After adapter clipping (trimmomatic v0.33), the reads were mapped against the human genome (bwa v0.7.12). Duplicate reads were removed (MarkDuplicates, Picard Tools v1.134), and SNP calling was conducted using VarScan v2.4.0 restricted to the regions targeted by the Nextera Rapid Capture Exome Kit. Finally, snpEff/snpSift v4.2 was applied for SNP effect prediction and SNP annotation. As no healthy control tissue was available for comparison, a SNP was defined as a difference between the sequenced sample and the reference genome (hg19). An important task in the context of tumor analysis is the detection of LOH (loss of heterozygosity). For that purpose, the number of SNPs with variant frequencies ≥0.95 or ≤0.05 were compared between bulk tumor and isolated tumor cells. In three out of four tumor specimens (OvCa_1, OvCa_3, OvCa_3_Met), a much higher number of SNPs fulfilling this selection criterion was detected in the isolated tumor cells compared to bulk tumor (A) indicating an improved detectability of LOH after tumor cell isolation. Figure B exemplifies this finding in tumor sample OvCa_3 for a deleterious SNP (ENST00000413465:c.112C>T p.Q38, COSM236889, not in dbSNP) causing a stop gain within a subset of transcript variants of the TP53 gene. While a variant frequency in the bulk tumor of 57% suggested a heterozygous SNP, an LOH event was indicated in the isolated tumor cells (97%) and the CD133+ cells (96%).

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