Handling, Storage, and Preparation of Human Tissues

Lynn G. Dressler1, Dan Visscher2

1 University of North Carolina, Chapel Hill, North Carolina, 2 Harper Hospital, Detroit, Michigan
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 5.2
DOI:  10.1002/0471142956.cy0502s01
Online Posting Date:  May, 2001
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Abstract

Human tissue for flow cytometry must be prepared as an adequate single‐cell suspension. The appropriate methods for tissue collection, transport, storage, and dissociation depend on the cell parameters being measured and the localization of the markers. This unit includes a general method for collecting and transporting human tissue and preparing a tissue imprint. Protocols are supplied for tissue disaggregation by either mechanical or enzymatic means and for preparation of single‐cell suspensions of whole cells from fine‐needle aspirates, pleural effusions, abdominal fluids, or other body fluids. Other protocols detail preparation of intact nuclei from fresh, frozen, or paraffin‐embedded tissue. Support protocols cover fixation, cryospin preparation, cryopreservation, and removal of debris. Keywords: flow cytometry; human tissue; tissue imprint; single‐cell suspension preparation; tissue disaggregation; paraffin‐embedded tissue

     
 
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Table of Contents

  • Basic Protocol 1: Collection and Transport of Samples to the Flow Cytometry Laboratory
  • Support Protocol 1: Preparing A Tissue Imprint
  • Basic Protocol 2: Mechanical Disaggregation of Whole Cells from Fresh Human Tissue
  • Alternate Protocol 1: Enzymatic Disaggregation of Whole Cells from Fresh Human Tissue
  • Basic Protocol 3: Preparation of Cells from Fine‐Needle Aspirates
  • Alternate Protocol 2: Preparation of Cells from Pleural Effusions Abdominal Fluids, and Other Fluids
  • Support Protocol 2: Ethanol Fixation of Cell Suspensions
  • Support Protocol 3: Preparation of Cytospin Slides from Cell Suspensions
  • Support Protocol 4: Freezing Dissociated Single Cells
  • Basic Protocol 4: Isolation of Nuclei from Fresh or Frozen Tissue
  • Alternate Protocol 3: Isolation of Nuclei from Paraffin‐Embedded Tissue
  • Support Protocol 5: Removing Debris from Suspensions of Nuclei
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Collection and Transport of Samples to the Flow Cytometry Laboratory

  Materials
  • Growth medium (e.g., MEM or RPMI 1640) containing 5% (v/v) heat‐inactivated FBS ( appendix 2A) or Hanks' balanced salt solution (HBSS; appendix 2A)
  • Liquid nitrogen or isopentane bath
  • Sample containers (e.g., 50‐ml conical centrifuge tubes or cryovials), sterile
  • Curved and straight forceps, sterile
  • Cryovials
  • Additional reagents and equipment for preparing a tissue imprint (see protocol 2)

Support Protocol 1: Preparing A Tissue Imprint

  Materials
  • Tissue specimen, freshly collected
  • 95% ethanol
  • Forceps
  • Glass slides
  • Coplin jar
  • Papanicolaou staining set or hematoxylin‐eosin staining series

Basic Protocol 2: Mechanical Disaggregation of Whole Cells from Fresh Human Tissue

  Materials
  • Tumor tissue, freshly obtained (see protocol 1)
  • Growth medium (e.g., RPMI 1640 medium, Life Technologies)
  • FBS, heat‐inactivated (Life Technologies or appendix 2A)
  • 0.4% (w/v) trypan blue (Sigma)
  • Gauze, sterile
  • No. 22 scalpel blades and handle
  • 100‐mm petri dishes
  • 80‐µm metal‐mesh sieve (EC Apparatus)
  • 15‐ and 50‐ml conical plastic centrifuge tubes
  • IEC CL centrifuge and swinging bucket rotor (or equivalent)
  • Additional reagents and equipment for counting cells ( appendix 3A) and assessing viability ( appendix 3B), and fixing cell suspension (see protocol 7)
NOTE: This protocol should be carried out in biological safety cabinet.

Alternate Protocol 1: Enzymatic Disaggregation of Whole Cells from Fresh Human Tissue

  • Trypsin
  • Collagenase type II
  • DNAse I
  • Additional reagents and equipment for counting cells ( appendix 3A) and assessing viability ( appendix 3B), and fixing cell suspension (see protocol 7)

Basic Protocol 3: Preparation of Cells from Fine‐Needle Aspirates

  Materials
  • Needle or bone marrow aspirate in a syringe or sterile container
  • Growth medium (e.g., MEM, HBSS, or RPMI 1640) containing 5% (v/v) heat‐inactivated FBS (Life Technologies or appendix 2A)
  • Hemolyzing reagent: 3.6% (w/v) NaCl in H 2O, cold
  • Heparin
  • 15‐ml or 50‐ml conical centrifuge tubes
  • Beckman RJ‐6 centrifuge and TH‐4 rotor (or equivalent)
  • Forceps, sterile
  • Additional reagents and equipment for counting cells ( appendix 3A)

Alternate Protocol 2: Preparation of Cells from Pleural Effusions Abdominal Fluids, and Other Fluids

  • Pleural effusion, abdominal fluid, or other fluid
  • 25‐ml pipet and bulb, sterile
  • 15‐ or 50‐ml centrifuge tubes or 250‐ml centrifuge bottles, sterile
  • Additional reagents and equipment for counting cells ( appendix 3B)

Support Protocol 2: Ethanol Fixation of Cell Suspensions

  Materials
  • Whole cell suspension (see protocol 3 or protocol 53 or protocol 4 or protocol 62)
  • 70% ethanol, 4°C
  • 15‐ml centrifuge tube

Support Protocol 3: Preparation of Cytospin Slides from Cell Suspensions

  Materials
  • PBS (see appendix 2A)
  • Ethanol‐fixed single‐cell suspension (see protocol 7)
  • 0.1% (w/v) bovine serum albumin (BSA, Baxter) in PBS
  • Leishman or Papanicolaou stain (Sigma)
  • 12 × 75‐mm tube
  • Glass slides
  • Cytocentrifuge (Shandon), slide chambers, filter cards

Support Protocol 4: Freezing Dissociated Single Cells

  Materials
  • Whole cell suspension (see protocol 3 or protocol 53 or protocol 4 or protocol 62), unfixed
  • Growth medium (e.g., RPMI, MEM, Medium 199) containing 5% (v/v) heat‐inactivated FBS ( appendix 2A)
  • Cryopreservation solution (see recipe)
  • 1.25‐ml cryovials
  • 12 × 75–mm polystyrene tubes
  • Beckman RJ‐6 centrifuge and TH‐4 rotor

Basic Protocol 4: Isolation of Nuclei from Fresh or Frozen Tissue

  Materials
  • Tumor tissue, fresh or frozen
  • Growth medium (e.g., MEM or RPMI 1640) containing 5% (v/v) heat‐inactivated FBS ( appendix 2A), 4°C
  • Sample container, sterile and dry
  • Petri dish, disposable
  • Curved and straight forceps, sterile
  • Scalpel and no. 20 blades
  • 50‐ to 70‐µm nylon mesh (Tetko)
  • 15‐ml conical centrifuge tube
  • Beckman RJ‐6 centrifuge and TH‐4 rotor (or equivalent)
  • Additional reagents and equipment for counting cells ( appendix 3A)

Alternate Protocol 3: Isolation of Nuclei from Paraffin‐Embedded Tissue

  Materials
  • Formalin‐fixed paraffin‐embedded tissue
  • Hematoxylin‐eosin or Diff‐Qwik stain
  • Xylene or Histoclear
  • Ethanol series: 100%, 95%, 70% and 50%
  • Pepsin solution: 0.5% (w/v) bovine pancreatic pepsin in 0.9% (w/v) NaCl
  • Growth medium (e.g., MEM, RPMI, HBSS) containing 5% (v/v) heat‐inactivated FBS ( appendix 2A)
  • Propidium iodide buffer (see recipe)
  • Microtome (adjustable, to cut 50‐µm and 4‐µm sections)
  • Glass slides
  • 13 × 100–mm glass screw top tubes
  • 50‐µm nylon mesh (Tetko4)
  • Additional reagents and equipment for counting cells ( appendix 3A)

Support Protocol 5: Removing Debris from Suspensions of Nuclei

  Materials
  • 1.75 M sucrose in PBS ( appendix 2A)
  • 1.5 M sucrose in PBS ( appendix 2A)
  • Nuclear suspension from fresh, frozen, or paraffin‐embedded tissue, filtered (see protocol 10 or protocol 11)
  • Growth medium containing 5% (v/v) heat‐inactivated FBS ( appendix 2A)
  • 12 × 75–mm centrifuge tube
  • Beckman RJ‐6 centrifuge and TH‐4 rotor (or equivalent)
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Figures

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Literature Cited

Literature Cited
   Bijman, J., Wagener, D.J., van Rennes, H., Wessles, J.M.C., and van den Brock, P. 1985. Flow cytometric evaluation of cell dispersion from human head and neck tumors. Cytometry 6:334‐341.
   Costa, A., Silvestrini, R., Del Bino, G., and Motta, R. 1987. Implications of disaggregation procedures on biological presentation of human solid tumors. Cell Tissue Kinet. 20:171‐180.
   Crissman, J.D., Zarbo, R.J., Niebylski, C.D., Corbett, T., and Weaver, D. 1988. Flow cytometric DNA analysis of colon adenocarcinomas: A comparative study of preparatory techniques. Mod. Pathol. 1:198‐204.
   Dressler, L.G. 1993. DNA flow cytometry measurements as surrogate endpoints in chemoprevention trials: Clinical, biological, and quality control considerations. J. Cell. Biochem. 17G:212‐218.
   Dressler, L.G. and Bartow, S.A. 1989. DNA flow cytometry in solid tumors: Practical aspects and clinical applications. Semin. Diagn. Pathol. 6:55‐82.
   Dressler, L.G. and Seamer, L.C. 1994. Controls, standards, and histogram interpretation in DNA flow cytometry. In Methods in Cell Biology, Vol. 41, 2nd ed. (Z. Darzynkiewicz, J.P. Robinson, and H.A. Crissman, eds.) pp. 241‐262. Academic Press, San Diego.
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   Hedley, D.W. 1994. DNA analysis from paraffin‐embedded blocks. In Methods in Cell Biology, Vol. 41, 2nd ed. (Z. Darzynkiewicz, J.P. Robinson, and H.A. Crissman, eds.) pp. 231‐240. Academic Press, San Diego.
   Hedley, D.W., Friedlander, M.L., and Taylor, I.W. 1983. Method for analysis of cellular DNA content of paraffin‐embedded pathological material using flow cytometry. J. Histochem. Cytochem. 31:1333‐1335.
   Hitchcock, C.L., Ensley, J.F., and Zalupski, M. 1996. Processing of solid tumors for DNA analysis by flow cytometry. In Basic and Clinical Applications of Flow Cytometry—Proceedings of the 24th Annual Detroit Cancer Symposium (F.A. Valeriate, A. Nakeff, and M. Valdivieso, eds.) pp. 159‐178. Kluwer Academic Publishers, Boston.
   König, J.J., van Dongen, J.W., and Schröder, F.H. 1993. Preferential loss of abnormal prostate carcinoma cells by collagenase treatment. Cytometry 14:805‐810.
   McDivitt, R.W., Stone, K.R., and Meyer, J.S. 1984. A method for dissociation of viable human breast cancer cells that produces flow cytometric kinetic information similar to that obtained by thymidine labelling. Cancer Res. 44:2628‐2633.
   Shankey, T.V., Rabinovitch, P.S., Bagwell, B., Bauer, K.D., Duque, R.E., Hedley, D.W., Mayall, B.H., and Wheeless, L. 1993. Guidelines for implementation of clinical DNA cytometry. Cytometry 14:472‐477.
   Visscher, D.W. and Crissman, J.D. 1994. Dissociation of intact cells from tumors and normal tissues. In Methods in Cell Biology, Vol 41: Flow Cytometry, 2nd ed. Part A. (Z. Darzynkiewicz, J.P. Robinson, and H.A. Crissman, eds.) pp 1‐13. Academic Press, San Diego.
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