Mechanical Dissociation: A Method to Obtain Viable Cells from a Tissue

Protocol

1. Preparation of the Tissue Homogenate

1. Dissect resected tissues (malignant and normal tissue resected from the operating room) are in the pathology lab by trained personnel for immediate pickup. Tumor, NANT (taken the furthest distance from the tumor as possible) and normal tissue fragments are routinely processed within 1 - 3 hr of surgical excision in a BSL2 laboratory using standard biosafety procedures for human tissues. A flow chart of the protocol is illustrated in Figure 1.
2. Weigh all tissue fragments (normal, NANT and tumor) and measure the length, width, and height (length x width x height). This is an important step for the subsequent normalization of cell subpopulations, extracted RNA, etc.
   NOTE: The range of sample size is 100 to 10,000 mm³ with no fat if possible.
3. Imprint the tumor fragment on a glass slide for H&E staining to verify that the tissue is actually part of the tumor.
   1. Do this by pressing a glass microscope slide on the tumor fragment and applying gentle pressure with your fingers for a few seconds.
   2. Fix the slide with isopropanol for 2 min followed by a washing step in water. Counterstain the tissue for 30 sec with Mayer's hematoxylin.
   3. Wash the slide in six baths of water. Stain in Phloxine B 2% for 15 sec.
   4. Wash in one bath of water followed by four baths of isopropanol and finish with one bath of water.
   5. Incubate in isopropanol for 1 min and drain. Clear in two baths of xylene.
   6. Mount with xylene based mounting medium. Examine for tumor cellularity (Figure 2).
      NOTE: Mainly, tumor cells stick to the imprinted slide - the stromal, lymphoid or adipose cells rarely remain, leaving spaces between the tumor cells (Figure 2).
4. Place the tissue fragment in a small culture dish containing 1 ml of the chemically defined, serum-free hematopoietic cell medium (hereafter referred to as medium) at room temperature and dice it into small pieces (~1 - 2 mm²) using a sterile scalpel.
5. Transfer everything (tissue fragments + medium) to a mechanical dissociator C tube.
6. Rinse the Petri dish and scalpel with 2 ml of medium using a Pasteur pipette and add this to the C tube (volume of medium for dissociation = 3 ml).
7. Use the mechanical dissociator program A.01 for C tubes (the most gentle program) to homogenize the tissue fragments into a single cell suspension. Place the C tube in the apparatus and run the program twice in succession (one cycle = 25 sec).
   NOTE: This homogenization procedure has been established and validated for human breast tissue, other tumor or tissue types may need to use a different program and should be tested first.
8. Remove the C tube from the apparatus and decant the homogenate directly into a 40 μm cell strainer seated on a 50 ml tube. Using the same Pasteur pipette as in step 1.6, transfer any liquid remaining in the C tube to the cell strainer.
9. Transfer the filtered liquid into a 15 ml tube using a 1 ml micropipette tip. Temporarily keep the cell strainer and its 50 ml tube.
10. Rinse the C tube with an additional 3 ml of medium and transfer this, again using the same Pasteur pipette as in step 1.6, to the cell strainer still seated on the 50 ml tube. Squeeze a maximum amount of the residual liquid trapped in the unhomogenized tissue into the 50 ml tube by gently moving it around the strainer with a clean Pasteur pipette or 1 ml tip that is subsequently thrown away to avoid contaminating the eluate.
11. Place the cell strainer upside down on the original C tube and rinse with 3 ml of medium so that the unhomogenized tissue drops back into the C tube.
12. Re-homogenize as in step 1.7 for two cycles of the A.01 program.
13. Pour this second homogenate through the cell strainer seated on the 50 ml tube, rinse the C tube again with 3 ml medium (as in step 1.10) and transfer with the Pasteur pipette to the cell strainer seated on the 50 ml tube again squeezing a maximum amount of liquid from the residual connective tissue trapped in the cell strainer.
14. At this point, a volume of ~2.5 ml is in the 15 ml tube and ~9 ml in the 50 ml tube.

2. Separation of the Tissue Supernatant and Cells

1. Centrifuge the homogenates in the 15 ml and 50 ml tubes for 15 min at 600 x g at room temperature.
2. Decant the SN from the 15 ml tube into a clean tube and temporarily store at 4 °C. This supernatant = primary tumor, NANT or normal tissue SN (final volume 2.5 ml) is subsequently clarified and aliquoted prior to storage at -80 °C for future analyses (see below).
3. Discard the supernatant from the 50 ml tube.
4. Gently resuspend both cell pellets in a final volume of 1 ml medium. Briefly, first gently break the cell pellet in both tubes (by tapping the tube on a hard surface). Resuspend the loose cell pellet in the 50 ml with 500 µl of medium and transfer this cell suspension to the 15 ml tube to resuspend the second pellet. Repeat this step once with the second 500 µl of medium for maximum recovery of cells.
5. Transfer 10 µl of the cell suspension to a small tube, mix with 10 µl of trypan blue (dilution 1:1) and count the number of viable cells using a hemocytometer.
   NOTE: At this point a fraction of the cell suspension can also be analyzed by flow cytometry to evaluate cell size, granularity and if desired a limited number of subpopulation markers for more precise assessment of the relative cell distribution in the homogenate prior to extensive analysis or experimentation. All analyses by flow cytometry incorporate CD45 labeling for normalization of subpopulations.
6. Pellet the cells by centrifugation at 300 x g for 10 min at room temperature. The cells from the tumor, NANT, or normal tissue are now ready for further purification or analysis. These additional steps are best when performed on the same day as surgery.
   NOTE: For flow cytometric analysis but not cell sorting the residual red blood cells should be lysed after antibody labeling by adding 0.4 ml of red blood cell lysis buffer to the cell pellet, immediately vortexing for 1 sec and incubating a minimum of 10 min at room temperature (protected from light) before analysis.

3. Clarification of the Tissue Supernatant

1. Centrifuge the 1.5 ml tubes with tissue SN at 15,000 x g for 15 min at 4 °C.
2. Carefully remove the supernatant without touching or disturbing the pellet. Transfer to a clean tube (or tubes) depending upon the number and volume of aliquots desired.
3. Store the supernatant at -80 °C for future use.

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תוצאות

Figure 1: Protocol flow chart. Our procedure for processing fresh human tissues and some analytical approaches that can subsequently be used to assess lymphocytes infiltrating human tissues are shown.

Figure 2: H&E stained image of a breast tumor tissue imprint. This impr...

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Materials

Name	Company	Catalog Number	Comments
GentleMacs Dissociator	Miltenyi Biotec	130-093-235	BD Medimachine is somewhat equivalent
Centrifuge 5810 R	Eppendorf		or other standard table top centrifuge
Centrifuge 5417 R	Eppendorf		or other standard microcentrifuge
Esco Class II A2 Biosafety Cabinet	ESCO global		or other standard BSL2 hood
Inverted Microscope	Nikon eclipse TS100		or other microscope compatible for a hemacytometer
Bürker Chamber Marienfield		640210	or other standard hemacytometer
Navios Flow Cytometer	Beckman Coulter		or other flow cytometer (8-10 color recommended)
GentleMacs C-Tube	Miltenyi Biotec	130-096-344	BD Medimachine uses Filcon
Cell Culture Dish	Sarstedt	72,710	or other non-pyrogenic plasticware
Disposable Scalpel	Swann-Morton	510	or standard single use sterile scalpel
BD Cell Strainer 40 µm	Becton Dickinson	734-0002	or other non-pyrogenic plasticware
BD Falcon Tube 50 ml	Becton Dickinson	352070	or other non-pyrogenic plasticware
BD Falcon Tube 15 ml	Becton Dickinson	352097	or other non-pyrogenic plasticware
BD FACS Tube 5 ml	Becton Dickinson	352008	or other non-pyrogenic plasticware
Sterile Pasteur Pipette 5 ml	VWR	612-1685	or other non-pyrogenic plasticware
Microfuge Tube 1.5 ml	Eppendorf	7805-00	or other non-pyrogenic plasticware
X-Vivo 20	Lonza	BE04-448Q	serum-free medium recommended
Phosphate buffered saline	Lonza	BE17-516F	standard physiological PBS
Trypan blue	VWR	17942E	or other vital stain