Application protocol

Isolation of xenografted cells from tumors by depletion of mouse cells

During growth phase in vivo, tumor tissue xenografted in mouse model systems is vascularized and infiltrated by cells of murine origin. Such contamination makes molecular downstream analysis, like expression profiling or next-generation sequencing, challenging. In addition, culturing human tumor cells is frequently hampered by murine fibroblasts overgrowing target cells.

In this application protocol, we describe a time-saving workflow that overcomes these limitations and increases sensitivity of downstream analyses. Xenograft tumor tissue is dissociated into a viable single-cell suspension and untouched human tumor cells are 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 depletion of mouse cells

  • Mouse Cell Depletion Kit (# 130-104-694)
  • QuadroMACS™ Starting Kit (LS) (# 130-091-051)
  • 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. 
    ▲ Note: 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. 
  • Pre-Separation Filters (70 µm) (# 130-095-823)
  • (Optional) Fluorochrome-conjugated antibodies for flow cytometry analysis, e.g., CD326 (EpCAM)-PE. Learn more about our antibodies and dyes.
  • (Optional) Propidium Iodide Solution (# 130-093-233) or 7-AAD for flow cytometry exclusion of dead cells without fixation
  • (Optional) Labeling Check Reagent conjugated to, e.g., APC to evaluate purity of sorted cells

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 xenograft 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

Human tumor cells are isolated by depleting the mouse cells using the Mouse Cell Depletion Kit and LS Columns. Follow the protocol from the kit data sheet.

Download the data sheet

Mouse Cell Depletion Kit


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 human tumor cells from xenograft tumors. The procedure is based on the comprehensive depletion of mouse cells from human tumor xenografts by magnetic cell separation.

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Flow cytometry analysis of purity of isolated human tumor cells after mouse cell depletion.  The original cell fraction (bulk tumor) and the negative cell fraction from the depletion (isolated human tumor cells) were labeled with a pan-mouse antibody cocktail and an antibody against human CD326 (EpCAM) and analyzed by flow cytometry. Over 99% of the contaminating mouse cells were eliminated in 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 fraction (left) and tumor cell fraction after mouse cell depletion (right) 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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Positive influence of non-tumor cell depletion on the quality of next-generation sequencing data. DNA from bulk tumor or isolated tumor cells of three different xenograft models derived from human kidney, lung, and bladder cancer were 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 cycles, Illumina) was utilized to generate 75-bp paired-end reads. As the capture oligonucleotides used for targeted enrichment of protein-coding sequences were designed based on the human genome, an initial pre-enrichment of DNA fragments of human origin from the mixture of mouse and human cells was expected. To assess the number of capture oligonucleotides that might cross-hybridize with mouse genomic DNA, BLAST searches of each single Nextera probe against mouse genome were conducted and the resulting alignment parameters were used to determine possible cross-hybridization. Depending on the selection thresholds (alignment length, no. of mismatches, no. of gaps), a cross-reactivity of 5–10% of capture probes with mouse transcripts was predicted (data not shown). (A) A significant increase (p < 0.05) in cluster density (not shown) as well as an average increase in read counts of 33% was observed for the samples depleted of mouse cells, indicating improved sample quality. A corresponding strong reduction of debris and dead cells upon mouse cell depletion was also apparent in flow cytometry analysis (data not shown). (B) After adapter clipping (trimmomatic v0.32), reads of all samples were mapped against human and mouse genomes (bwa v0.7.12) and putative origin was determined based on the respective alignment parameters (LINUX shell, command-line Perl). (C) Detailed read assignment for bulk tumor and isolated human tumor cells derived from the bladder cancer xenograft. An average of 12% of reads derived from bulk tumor samples was attributed to mouse cells. This amount could be reduced to 0.3% by prior depletion of mouse cells. On average, 15% of the mouse-derived reads mapped erroneously to the human genome (1.9% of total reads) in the bulk tumor samples, demonstrating a strong positive influence of mouse cell depletion (0.04% of total reads erroneously mapped to human genome) on downstream analyses.

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