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Protocol
Tissue Dissociation for Metastasis Studies Farhia Kabeer1,3,4 and Katrina Podsypanina1,2,3 1
Institut de Recherches Cliniques de Montréal, Quebec H2W 1R7, Canada; 2Pathologie et Biologie Cellulaire, Université de Montréal, Quebec H3T 1J4, Canada; 3Department of Medicine, Experimental Medicine Division, McGill University, Montréal, Quebec H3A 13A, Canada
The main requirement for most metastasis-related applications is the conversion of solid tissue into a single-cell suspension. In theory, this suspension represents the diversity of cells present in the tissue, whether malignant or benign. We have found that cell viability, as measured by trypan blue staining or fluorescence-activated cell sorting (FACS), is critical for evaluating the success of the tissue-dissociation procedure. The recommended goal is at least 70% cell viability.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents If dissociating fragments of tumors that have formed in the presence of doxycycline (as in the Tet-On [rtTA] system), add doxycycline to all reagents to a final concentration of 1 µg/mL. Alternatively, if using the Tet-Off (tTA) system, ensure that the fetal bovine serum (FBS) and all reagents are certified “tetracycline-free.”
Complete medium for metastasis studies Digestion solution for tissue dissociation DMEM/F12 medium DNase I (Sigma-Aldrich; amplification grade; 1000 U/mL or 0.5 mg/mL) HEPES buffer (1 M) PBS with Ca2+ and Mg2+ (D-PBS) Phosphate-buffered saline (PBS) (10×; pH 7.4) This PBS does not contain Ca2+ or Mg2+.
RBC lysis buffer for tissue dissociation Trypan blue stain (0.4%) Trypsin-EDTA solution (HBSS containing 0.25% trypsin and 0.53 mM EDTA; Wisent 325-043-EL) Equipment
Alcohol swabs Cell-culture incubator (37˚C/5% CO2) (optional; see Steps 15–17) 4
Correspondence: [email protected]
© 2016 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot078329
160
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Tissue Dissociation for Metastasis Studies
Centrifuge Conical tubes (15 mL) Hemocytometer (e.g., Reichert Bright-Line) Needles (18, 21, and/or 22 gauge; see Steps 2 and 5) Nylon mesh filter (40-µm pore size) Pasteur pipettes Pipettes (5 mL) Scissors (10 cm) Syringes (5 and/or 10 mL; see Steps 2 and 5) Tissue-culture plate (six well) (optional; see Steps 15–17) Tissue forceps Transfer pipettes Water bath at 37˚C METHOD Dissociating the Tissue
1. Thaw an appropriate amount of digestion solution and warm it to 37˚C. Use one 5-mL aliquot per mouse tissue sample (i.e., one lung, eight mammary glands, or one 10 mm × 5 mm tumor fragment). 2. Clean the scissors and tissue forceps with alcohol swabs. Dissect and remove the tissue to be digested.
• • •
To obtain normal mammary cells, remove the thoracic and inguinal mammary glands from a 6- to 10-wk-old virgin female mouse. Excise the inguinal lymph node from the middle of each inguinal mammary gland. To obtain tumor cells from a primary tumor site, remove no more than a 10 mm × 5 mm fragment of tumor tissue from a transgenic mouse. To obtain tumor cells from a metastatic site in the lung, perfuse the lung with D-PBS: Slice open the left renal artery and, using a 21-gauge needle and a 5-mL syringe, pass 3–5 mL of D-PBS through the right ventricle of the heart.
3. Place eight mammary glands, an entire lung, or one 10 mm × 5 mm tumor fragment into a 15-mL conical tube. 4. Use scissors to cut the tissues into small pieces. Mince the tissues to the consistency of paste and continue to cut until the pieces are small enough to pass through a 5-mL pipette. Add 5 mL of digestion solution to each tissue sample, and place the tubes into a 37˚C water bath.
5. Incubate the tubes at 37˚C for 1.5–2.5 h with trituration.
•
For mammary glands and tumors, shake the tube and triturate the tissue with a transfer pipette every 15 min until no macroscopic fragments are visible.
•
For lung tissue, after the first hour, triturate every 15 min with a 10-mL syringe, beginning with an 18-gauge needle and finishing with a 22-gauge needle. If within 1.5 h gross tissue chunks are still abundant, this serves as an indication that smaller tissue fragments should be used in subsequent preparations. See Troubleshooting and Discussion.
6. Add 10 mL of DMEM/F12 medium. Centrifuge the tissue at 1000 rpm (233g) for 10 min at 25˚C. Aspirate the supernatant, and resuspend the cells in 1 mL of trypsin–EDTA solution for 2 min at 25˚C with occasional shaking (or pipette them up and down with a Pasteur pipette) to ensure that the enzyme can access all of the tissue. See Troubleshooting.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot078329
161
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F. Kabeer and K. Podsypanina
7. Add DNase I to trypsinized cells (final concentration 200 U/mL or 0.1 mg/mL), mix with a pipette, and incubate for an additional 3 min at 25˚C.
This is a critical step; in the absence of DNase I, the trypsinized tissue will not pass through the filter in Step 9.
8. Add 9 mL of complete medium. This is a critical step; in the absence of serum, the trypsin will continue to digest the tissue and decrease the cell viability.
9. Filter the mixture through a 40-μm nylon mesh filter. Centrifuge at 1000 rpm (233g) for 10 min at 25˚C. See Troubleshooting.
10. Aspirate the supernatant. Resuspend the cell pellet in 3 mL of RBC lysis buffer, and incubate for 1 min at 25˚C. After 1 min, add 10 mL of D-PBS and centrifuge at 1000 rpm (233g) for 10 min at 25˚C.
11. Aspirate the supernatant. Resuspend the cell pellet in 1–3 mL of D-PBS containing 10 mM HEPES (or DMEM/F12 medium). At this point, the cells can be counted (see Steps 12–14), sorted by FACS, plated for primary culture and viral transduction (for mammary cells, see Steps 15–17), transferred to new recipients by injection, or stored for molecular assays at −80˚C or in liquid nitrogen for 4 yr.
Counting the Cells
12. Trypan blue viability is a dye exclusion method that utilizes membrane integrity to identify dead cells (non-viable). Healthy (viable) cells normally do not take up the dye for a short while. Dilute the 0.4% stock solution of trypan blue 1/10 in 1× PBS. Mix 5 µL of the final cell suspension with 45 µL of the 0.04% trypan blue solution. Place 10 µL of the mix on a hemocytometer. 13. Count the cells in four 16-square quadrants (viable and nonviable separately). To calculate percentage of viability use viable cell count × 100 = % viability. nonviable cell count Primary epithelial cells often stay in small clumps of two to 12 cells. These are different than the larger 40- to 100-cell organoids present after incomplete digestion. Cells in the clumps should be counted individually. If we need to stain with antibodies after, then nonviable cells should be taken into account as they can disturb estimated antibody dilution.
14. Average the cell counts. Multiply the average by 100,000 to estimate number of cells per milliliter. Approximately 10 million total cells can regularly be collected per eight mammary glands or 10 mm × 5 mm tumor fragment. See Troubleshooting.
(Optional) Mammary Cell Culture and Viral Transduction
15. Dilute the mammary cells with complete medium.
•
If using unfractionated primary mammary cells, dilute the cells to 1 × 106 cells/mL in complete medium.
•
If using FACS-sorted cells, dilute the cells to 5 × 105 cells/mL in complete medium.
16. Plate 3 mL of the cell suspension into one well of a six well plate. Swirl the plate so that the mixture is evenly distributed, and place the plate into a 37˚C/5% CO2 cell-culture incubator. See Troubleshooting.
17. On the next day, transduce the cells with vectors produced in Protocol: Murine Stem Cell–Based Retrovirus Production for Marking Primary Mouse Mammary Cells for Metastasis Studies (Beverly and Podsypanina 2014). 162
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot078329
Downloaded from http://cshprotocols.cshlp.org/ at SERIALS/BIOMED LIB0175B on February 8, 2016 - Published by Cold Spring Harbor Laboratory Press
Tissue Dissociation for Metastasis Studies
TROUBLESHOOTING Problem (Step 5): The sample is not easily drawn through the syringe. Solution: It is possible to accelerate digestion by triturating with needles of decreasing gauge; however,
this often leads to a decrease in cell viability. Apply mild pressure, and keep in mind the effect on cell viability. Problem (Step 6): The sample does not pellet completely. Solution: Incomplete pelleting may occur when residual fat tissue is present. Draw the mixture up and
down through an 18-gauge needle attached to a 10-mL syringe. Do this up to five times. Repeat with 20- and 22-gauge needles. Problem (Step 9): The sample does not filter easily. Solution: Use the syringe plunger to grind the cell mass against the surface of the filter. Flush with final
concentration of 0.1mg/mL DNase I followed by complete medium. Problem (Step 14): The yield of viable cells is low. Solution: Viable cells may have been lost in undigested pieces, or there may be an increased number of
dead cells because of a lengthy digest of a relatively small sample. For the former, reduce the amount of tissue to be digested; for the latter, reduce the length of the digestion. Problem (Step 16): The plating efficiency is low. Solution: Postsorting cell numbers are often low, especially from colonized organs. If necessary, cell
seeding can be performed in wells of 12-well, 24-well, or 96-well plates. Alternatively, the cell number plated can be increased to 106 per well. If cells died in the sorting process, be sure to transfer cells into complete medium immediately after sorting.
DISCUSSION
Many tissue-dissociating protocols for both malignant and benign tissues are available in the literature (Zhang et al. 2008; Lukacs et al. 2010; Mroue and Bissell 2013). Although most are a combination of mechanical dissociation with enzymatic digestion, the precise technique, timing of the procedure, and the enzymes used vary widely even for the same tissue (Shackleton et al. 2006; Stingl et al. 2006; Smalley 2010). We find that the efficacy of dissociation is a function of enzyme, tissue, and agitation (i.e., it depends on the enzyme:tissue ratio and the intensity of tissue mincing and agitation). Tissues of different origin have different resistance to enzymatic digestion, and the appropriate enzyme for a given tissue should be determined empirically. However, in studies of metastatic processes it is often necessary to isolate normal, malignant, primary, and colonizing cells under equivalent conditions. For those who are performing tissue dissociation for the first time, we recommend reviewing several available protocols and beginning with the understanding that several optimization steps will be necessary to adapt the protocol for your experimental needs. Single-cell dissociation of solid tissues prepares the investigator to analyze entire populations of tumor or tissue cells. In principle, this procedure preserves the diversity inherent in complex tissues and even in tumor cells themselves (Gerlinger et al. 2012), which is indispensable for proper measurement of the metastatic proclivity of an entire tumor. Our protocol, when applied to colonized organs, consistently yields functionally competent cells (Podsypanina et al. 2008). However, we cannot exclude the possibility that a specific subset of cells in a given tissue or tumor is exquisitely vulnerable to dissociation procedures and thus will be consistently underrepresented in studies employing these procedures. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot078329
163
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F. Kabeer and K. Podsypanina
RECIPES Complete Medium for Metastasis Studies
50 mL fetal bovine serum, tetracycline free (FBS-TF) 5 mL penicillin-streptomycin stock solution (10,000 U/mL penicillin and 10,000 µg/mL streptomycin) 450 mL DMEM/F12 medium Filter-sterilize, aliquot, and store in a dark bottle. Keep for up to 3 wk at 4˚C or for up to 2 mo at −20˚C. Digestion Solution for Tissue Dissociation
10 mL collagenase/hyaluronidase in DMEM (10×; StemCell Technologies 07912) 90 mL complete medium for metastasis studies Filter-sterilize using a 0.22-μm filter. Aliquot and store for up to 2 mo at −20˚C. PBS (10×; pH 7.4)
320 g NaCl 8 g KCl 57.6 g Na2HPO4 9.6 g KH2PO4 Dissolve in 3 L of distilled H2O. Adjust the pH to 7.4. Bring to 4 L with distilled H2O. Autoclave. Store at room temperature for up to 1 yr.
PBS with Ca2+and Mg2+ (D-PBS)
0.1 g CaCl2 0.2 g KCl 0.1 g MgCl2 · 6H2O 8 g NaCl 2.16 g Na2HPO4 Add ddH2O to a final volume of 1 L and filter-sterilize. Store at 25˚C for up to 12 mo. RBC Lysis Buffer for Tissue Dissociation
0.0185 g EDTA (tetrasodium salt dihydrate) 0.5 g KHCO3 4.13 g NH4Cl 500 mL double-distilled H2O Mix in a large (≥500-mL) flask and autoclave. Store for up to 6 mo at room temperature or at 4˚C. Always use at room temperature.
REFERENCES Beverly LJ, Podsypanina K. 2014. Murine stem cell–based retrovirus production for marking primary mouse mammary cells for metastasis studies. Cold Spring Harb Protoc doi: 10.1101/pdb.prot078337. Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, Martinez P, Matthews N, Stewart A, Tarpey P, et al. 2012. Intratumor
164
heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 366: 883–892. Lukacs RU, Goldstein AS, Lawson DA, Cheng D, Witte ON. 2010. Isolation, cultivation and characterization of adult murine prostate stem cells. Nat Protoc 5: 702–713.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot078329
Downloaded from http://cshprotocols.cshlp.org/ at SERIALS/BIOMED LIB0175B on February 8, 2016 - Published by Cold Spring Harbor Laboratory Press
Tissue Dissociation for Metastasis Studies Mroue R, Bissell MJ. 2013. Three-dimensional cultures of mouse mammary epithelial cells. Methods Mol Biol 945: 221–250. Podsypanina K, Du YC, Jechlinger M, Beverly LJ, Hambardzumyan D, Varmus H. 2008. Seeding and propagation of untransformed mouse mammary cells in the lung. Science 321: 1841–1844. Shackleton M, Vaillant F, Simpson KJ, Stingl J, Smyth GK, AsselinLabat ML, Wu L, Lindeman GJ, Visvader JE. 2006. Generation of a functional mammary gland from a single stem cell. Nature 439: 84–88.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot078329
Smalley MJ. 2010. Isolation, culture and analysis of mouse mammary epithelial cells. Methods Mol Biol 633: 139–170. Stingl J, Eirew P, Ricketson I, Shackleton M, Vaillant F, Choi D, Li HI, Eaves CJ. 2006. Purification and unique properties of mammary epithelial stem cells. Nature 439: 993–997. Zhang M, Behbod F, Atkinson RL, Landis MD, Kittrell F, Edwards D, Medina D, Tsimelzon A, Hilsenbeck S, Green JE, et al. 2008. Identification of tumor-initiating cells in a p53–null mouse model of breast cancer. Cancer Res 68: 4674–4682.
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Protocol
Tissue Dissociation for Metastasis Studies Farhia Kabeer1,3,4 and Katrina Podsypanina1,2,3 1
Institut de Recherches Cliniques de Montréal, Quebec H2W 1R7, Canada; 2Pathologie et Biologie Cellulaire, Université de Montréal, Quebec H3T 1J4, Canada; 3Department of Medicine, Experimental Medicine Division, McGill University, Montréal, Quebec H3A 13A, Canada
The main requirement for most metastasis-related applications is the conversion of solid tissue into a single-cell suspension. In theory, this suspension represents the diversity of cells present in the tissue, whether malignant or benign. We have found that cell viability, as measured by trypan blue staining or fluorescence-activated cell sorting (FACS), is critical for evaluating the success of the tissue-dissociation procedure. The recommended goal is at least 70% cell viability.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents If dissociating fragments of tumors that have formed in the presence of doxycycline (as in the Tet-On [rtTA] system), add doxycycline to all reagents to a final concentration of 1 µg/mL. Alternatively, if using the Tet-Off (tTA) system, ensure that the fetal bovine serum (FBS) and all reagents are certified “tetracycline-free.”
Complete medium for metastasis studies Digestion solution for tissue dissociation DMEM/F12 medium DNase I (Sigma-Aldrich; amplification grade; 1000 U/mL or 0.5 mg/mL) HEPES buffer (1 M) PBS with Ca2+ and Mg2+ (D-PBS) Phosphate-buffered saline (PBS) (10×; pH 7.4) This PBS does not contain Ca2+ or Mg2+.
RBC lysis buffer for tissue dissociation Trypan blue stain (0.4%) Trypsin-EDTA solution (HBSS containing 0.25% trypsin and 0.53 mM EDTA; Wisent 325-043-EL) Equipment
Alcohol swabs Cell-culture incubator (37˚C/5% CO2) (optional; see Steps 15–17) 4
Correspondence: [email protected]
© 2016 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot078329
160
Downloaded from http://cshprotocols.cshlp.org/ at SERIALS/BIOMED LIB0175B on February 8, 2016 - Published by Cold Spring Harbor Laboratory Press
Tissue Dissociation for Metastasis Studies
Centrifuge Conical tubes (15 mL) Hemocytometer (e.g., Reichert Bright-Line) Needles (18, 21, and/or 22 gauge; see Steps 2 and 5) Nylon mesh filter (40-µm pore size) Pasteur pipettes Pipettes (5 mL) Scissors (10 cm) Syringes (5 and/or 10 mL; see Steps 2 and 5) Tissue-culture plate (six well) (optional; see Steps 15–17) Tissue forceps Transfer pipettes Water bath at 37˚C METHOD Dissociating the Tissue
1. Thaw an appropriate amount of digestion solution and warm it to 37˚C. Use one 5-mL aliquot per mouse tissue sample (i.e., one lung, eight mammary glands, or one 10 mm × 5 mm tumor fragment). 2. Clean the scissors and tissue forceps with alcohol swabs. Dissect and remove the tissue to be digested.
• • •
To obtain normal mammary cells, remove the thoracic and inguinal mammary glands from a 6- to 10-wk-old virgin female mouse. Excise the inguinal lymph node from the middle of each inguinal mammary gland. To obtain tumor cells from a primary tumor site, remove no more than a 10 mm × 5 mm fragment of tumor tissue from a transgenic mouse. To obtain tumor cells from a metastatic site in the lung, perfuse the lung with D-PBS: Slice open the left renal artery and, using a 21-gauge needle and a 5-mL syringe, pass 3–5 mL of D-PBS through the right ventricle of the heart.
3. Place eight mammary glands, an entire lung, or one 10 mm × 5 mm tumor fragment into a 15-mL conical tube. 4. Use scissors to cut the tissues into small pieces. Mince the tissues to the consistency of paste and continue to cut until the pieces are small enough to pass through a 5-mL pipette. Add 5 mL of digestion solution to each tissue sample, and place the tubes into a 37˚C water bath.
5. Incubate the tubes at 37˚C for 1.5–2.5 h with trituration.
•
For mammary glands and tumors, shake the tube and triturate the tissue with a transfer pipette every 15 min until no macroscopic fragments are visible.
•
For lung tissue, after the first hour, triturate every 15 min with a 10-mL syringe, beginning with an 18-gauge needle and finishing with a 22-gauge needle. If within 1.5 h gross tissue chunks are still abundant, this serves as an indication that smaller tissue fragments should be used in subsequent preparations. See Troubleshooting and Discussion.
6. Add 10 mL of DMEM/F12 medium. Centrifuge the tissue at 1000 rpm (233g) for 10 min at 25˚C. Aspirate the supernatant, and resuspend the cells in 1 mL of trypsin–EDTA solution for 2 min at 25˚C with occasional shaking (or pipette them up and down with a Pasteur pipette) to ensure that the enzyme can access all of the tissue. See Troubleshooting.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot078329
161
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F. Kabeer and K. Podsypanina
7. Add DNase I to trypsinized cells (final concentration 200 U/mL or 0.1 mg/mL), mix with a pipette, and incubate for an additional 3 min at 25˚C.
This is a critical step; in the absence of DNase I, the trypsinized tissue will not pass through the filter in Step 9.
8. Add 9 mL of complete medium. This is a critical step; in the absence of serum, the trypsin will continue to digest the tissue and decrease the cell viability.
9. Filter the mixture through a 40-μm nylon mesh filter. Centrifuge at 1000 rpm (233g) for 10 min at 25˚C. See Troubleshooting.
10. Aspirate the supernatant. Resuspend the cell pellet in 3 mL of RBC lysis buffer, and incubate for 1 min at 25˚C. After 1 min, add 10 mL of D-PBS and centrifuge at 1000 rpm (233g) for 10 min at 25˚C.
11. Aspirate the supernatant. Resuspend the cell pellet in 1–3 mL of D-PBS containing 10 mM HEPES (or DMEM/F12 medium). At this point, the cells can be counted (see Steps 12–14), sorted by FACS, plated for primary culture and viral transduction (for mammary cells, see Steps 15–17), transferred to new recipients by injection, or stored for molecular assays at −80˚C or in liquid nitrogen for 4 yr.
Counting the Cells
12. Trypan blue viability is a dye exclusion method that utilizes membrane integrity to identify dead cells (non-viable). Healthy (viable) cells normally do not take up the dye for a short while. Dilute the 0.4% stock solution of trypan blue 1/10 in 1× PBS. Mix 5 µL of the final cell suspension with 45 µL of the 0.04% trypan blue solution. Place 10 µL of the mix on a hemocytometer. 13. Count the cells in four 16-square quadrants (viable and nonviable separately). To calculate percentage of viability use viable cell count × 100 = % viability. nonviable cell count Primary epithelial cells often stay in small clumps of two to 12 cells. These are different than the larger 40- to 100-cell organoids present after incomplete digestion. Cells in the clumps should be counted individually. If we need to stain with antibodies after, then nonviable cells should be taken into account as they can disturb estimated antibody dilution.
14. Average the cell counts. Multiply the average by 100,000 to estimate number of cells per milliliter. Approximately 10 million total cells can regularly be collected per eight mammary glands or 10 mm × 5 mm tumor fragment. See Troubleshooting.
(Optional) Mammary Cell Culture and Viral Transduction
15. Dilute the mammary cells with complete medium.
•
If using unfractionated primary mammary cells, dilute the cells to 1 × 106 cells/mL in complete medium.
•
If using FACS-sorted cells, dilute the cells to 5 × 105 cells/mL in complete medium.
16. Plate 3 mL of the cell suspension into one well of a six well plate. Swirl the plate so that the mixture is evenly distributed, and place the plate into a 37˚C/5% CO2 cell-culture incubator. See Troubleshooting.
17. On the next day, transduce the cells with vectors produced in Protocol: Murine Stem Cell–Based Retrovirus Production for Marking Primary Mouse Mammary Cells for Metastasis Studies (Beverly and Podsypanina 2014). 162
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot078329
Downloaded from http://cshprotocols.cshlp.org/ at SERIALS/BIOMED LIB0175B on February 8, 2016 - Published by Cold Spring Harbor Laboratory Press
Tissue Dissociation for Metastasis Studies
TROUBLESHOOTING Problem (Step 5): The sample is not easily drawn through the syringe. Solution: It is possible to accelerate digestion by triturating with needles of decreasing gauge; however,
this often leads to a decrease in cell viability. Apply mild pressure, and keep in mind the effect on cell viability. Problem (Step 6): The sample does not pellet completely. Solution: Incomplete pelleting may occur when residual fat tissue is present. Draw the mixture up and
down through an 18-gauge needle attached to a 10-mL syringe. Do this up to five times. Repeat with 20- and 22-gauge needles. Problem (Step 9): The sample does not filter easily. Solution: Use the syringe plunger to grind the cell mass against the surface of the filter. Flush with final
concentration of 0.1mg/mL DNase I followed by complete medium. Problem (Step 14): The yield of viable cells is low. Solution: Viable cells may have been lost in undigested pieces, or there may be an increased number of
dead cells because of a lengthy digest of a relatively small sample. For the former, reduce the amount of tissue to be digested; for the latter, reduce the length of the digestion. Problem (Step 16): The plating efficiency is low. Solution: Postsorting cell numbers are often low, especially from colonized organs. If necessary, cell
seeding can be performed in wells of 12-well, 24-well, or 96-well plates. Alternatively, the cell number plated can be increased to 106 per well. If cells died in the sorting process, be sure to transfer cells into complete medium immediately after sorting.
DISCUSSION
Many tissue-dissociating protocols for both malignant and benign tissues are available in the literature (Zhang et al. 2008; Lukacs et al. 2010; Mroue and Bissell 2013). Although most are a combination of mechanical dissociation with enzymatic digestion, the precise technique, timing of the procedure, and the enzymes used vary widely even for the same tissue (Shackleton et al. 2006; Stingl et al. 2006; Smalley 2010). We find that the efficacy of dissociation is a function of enzyme, tissue, and agitation (i.e., it depends on the enzyme:tissue ratio and the intensity of tissue mincing and agitation). Tissues of different origin have different resistance to enzymatic digestion, and the appropriate enzyme for a given tissue should be determined empirically. However, in studies of metastatic processes it is often necessary to isolate normal, malignant, primary, and colonizing cells under equivalent conditions. For those who are performing tissue dissociation for the first time, we recommend reviewing several available protocols and beginning with the understanding that several optimization steps will be necessary to adapt the protocol for your experimental needs. Single-cell dissociation of solid tissues prepares the investigator to analyze entire populations of tumor or tissue cells. In principle, this procedure preserves the diversity inherent in complex tissues and even in tumor cells themselves (Gerlinger et al. 2012), which is indispensable for proper measurement of the metastatic proclivity of an entire tumor. Our protocol, when applied to colonized organs, consistently yields functionally competent cells (Podsypanina et al. 2008). However, we cannot exclude the possibility that a specific subset of cells in a given tissue or tumor is exquisitely vulnerable to dissociation procedures and thus will be consistently underrepresented in studies employing these procedures. Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot078329
163
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F. Kabeer and K. Podsypanina
RECIPES Complete Medium for Metastasis Studies
50 mL fetal bovine serum, tetracycline free (FBS-TF) 5 mL penicillin-streptomycin stock solution (10,000 U/mL penicillin and 10,000 µg/mL streptomycin) 450 mL DMEM/F12 medium Filter-sterilize, aliquot, and store in a dark bottle. Keep for up to 3 wk at 4˚C or for up to 2 mo at −20˚C. Digestion Solution for Tissue Dissociation
10 mL collagenase/hyaluronidase in DMEM (10×; StemCell Technologies 07912) 90 mL complete medium for metastasis studies Filter-sterilize using a 0.22-μm filter. Aliquot and store for up to 2 mo at −20˚C. PBS (10×; pH 7.4)
320 g NaCl 8 g KCl 57.6 g Na2HPO4 9.6 g KH2PO4 Dissolve in 3 L of distilled H2O. Adjust the pH to 7.4. Bring to 4 L with distilled H2O. Autoclave. Store at room temperature for up to 1 yr.
PBS with Ca2+and Mg2+ (D-PBS)
0.1 g CaCl2 0.2 g KCl 0.1 g MgCl2 · 6H2O 8 g NaCl 2.16 g Na2HPO4 Add ddH2O to a final volume of 1 L and filter-sterilize. Store at 25˚C for up to 12 mo. RBC Lysis Buffer for Tissue Dissociation
0.0185 g EDTA (tetrasodium salt dihydrate) 0.5 g KHCO3 4.13 g NH4Cl 500 mL double-distilled H2O Mix in a large (≥500-mL) flask and autoclave. Store for up to 6 mo at room temperature or at 4˚C. Always use at room temperature.
REFERENCES Beverly LJ, Podsypanina K. 2014. Murine stem cell–based retrovirus production for marking primary mouse mammary cells for metastasis studies. Cold Spring Harb Protoc doi: 10.1101/pdb.prot078337. Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, Martinez P, Matthews N, Stewart A, Tarpey P, et al. 2012. Intratumor
164
heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 366: 883–892. Lukacs RU, Goldstein AS, Lawson DA, Cheng D, Witte ON. 2010. Isolation, cultivation and characterization of adult murine prostate stem cells. Nat Protoc 5: 702–713.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot078329
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