Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Podsumowanie

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Streszczenie

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses¹.

Various strategies such as Staput^2,3, centrifugal elutriation¹, and flow cytometry (FC)^4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies.

It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium^6,7 - and lack cell clumps¹ . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones¹ . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types^1,8 .

The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting⁴, and ulterior gene expression studies⁹ .

Protokół

1. Preparation of Cell Suspensions

1. Sacrifice the specimen to be used following the recommendations of the specialized committees such as IACUC or equivalent (in Uruguay, National Commission for Animal Experimentation [CNEA]). In our case, an overdose of pentobarbital was administrated.
2. Dissect the testes following standard approved procedures and place them in a 96 mm glass Petri dish on ice, containing 10 ml of ice-cold DMEM supplemented with 10% fetal calf serum.
3. Remove the tunica albuginea and cut the decapsulated testes into square pieces of 2 - 3 mm on each side.
4. Those pieces are then processed in a Medimachine, an automated mechanical grinder where the tissue is disaggregated inside a disposable unit containing a perforated stainless-steel screen and a metal rotor. To do so, place 1 ml of cold supplemented DMEM and 4 - 5 of these pieces in a 50 μm disposable unit, switch on the disaggregator, and process for 50 sec following the simple instructions from the manufacturer.
5. Recover the resulting cell suspension from the disaggregation unit using a 3 - 5 ml syringe without needle.
6. Filter through a 50 μm nylon mesh, previously soaked with 0.5 ml supplemented DMEM.
7. Filter the suspension again using a soaked 25 μm nylon mesh, and place on ice.
8. Count in a Neubauer chamber and adjust cellular concentration to 1 - 2 x 10⁷ cells/ml with supplemented DMEM. At least 4 x 10⁷ cells/gram of testis material are usually obtained.
9. Finally, add NDA (2-naphthol-6,8-disulfonic acid, dipotassium salt) to a final concentration of 0.2% in order to prevent cell clumping.
10. Optional: check cell viability of the testicular cell suspensions with a commercially available viability kit for animal cells, following manufacturer's instructions.

2. Flow Cytometric Analysis

We have used a Becton-Dickinson FACSVantage flow cytometer equipped with a Coherent argon ion laser tuned to emit at 488 nm for the analysis of cells stained with high concentrations of propidium iodide (PI). (The issue of PI entrance into unfixed cells under stress has been addressed elsewhere^9,10).

1. For PI staining, add fluorochrome at a final concentration of 50 μg/ml to the cell suspension, and incubate for 10 min at 0 °C in the dark.
2. Laser power is set to 100 mW and a 575/26 band pass filter is used to collect PI-emitted fluorescence in FL2.
3. We perform FC measurements with a 70 μm nozzle. For sorting spermatocyte populations, set sorting mode in Normal-R or Normal-C, using 3 sorted drops as envelope. Keep sample and collection tubes at 3 - 4 °C by using a refrigeration unit. Adjust sample differential to analyze cells at a rate of 500 to 1,500 per second.
4. Use CellQuest software (BD) to analyze the following parameters: forward scatter (FSC-H); side scatter (SSC-H); total emitted fluorescence or pulse-area (FL2-A); and duration of fluorescence emission or pulse-width (FL2-W).

Alternatively, we have employed the vital dye Hoechst 33342 to a final concentration of 5 μg/ml and incubated for 10 min at 37 °C in the dark. Cell analysis was performed by means of a MoFlo Cytometer (DakoCytomation) equipped with a UV excitation wavelength laser operating at 25 mW and using a 70 μm nozzle. Data manipulation was performed with Summit v4.3 software (DakoCytomation).

Wyniki

An example of a well disaggregated cell suspension from rat testes prepared with the protocol described here is shown in Figure 1.

In comparison to enzymatic treatments^6,8 and to previously described mechanical disaggregation methods², the one presented here is much faster, involves less handling, is easily reproducible (not operator-dependant), and renders scarce cell debris (especially compared to other mechanical methods) and very few multinucleates (...

Dyskusje

The optimized method described here enables the preparation of cell suspensions from rodent testicular tissue in a very fast and reproducible way, avoiding enzyme and detergent treatment and maintaining good cell integrity and type proportions. The brevity of the procedure (the 15 min span includes testis dissection, tissue cutting, and processing), minimal handling involved, and absence of enzymatic treatments are some of the main advantages. All these would account for the good preservation of short life macromolecules...

Materiały

Name	Company	Catalog Number	Comments
Medimachine system	BD	340587
Medicon unit (50 μm)	BD	340591
Filcon unit (50 μm)	BD	340603
DMEM	Gibco	430-2100
Fetal calf serum	PAA	A11-151
NDA	Chemos BmbH	277081
H–chst 33342	Sigma-Aldrich	14533	Stock solution 5 mg/ml; used at a final concentration of 5 μg/ml
Propidium iodide	Sigma-Aldrich	287075	Stock solution 1 mg/ml; used at a final concentration of 50 μg/ml