The tissue in the medium is forcefully passed through a series of sieves of decreasing pore size until a desirable suspension of single cells or small tissue masses is produced, and the suspension can be directly diluted and cultured.

Operation method

Program 12.9 Mechanical Separation Using Screens

Principle

The tissue in the medium is forcefully passed through a series of sieves of decreasing pore size until a desirable suspension of single cells or small tissue masses is produced, and the suspension can be directly diluted and cultured.

Materials and Instruments

Growth media Tweezers Sieves Picot dishes Scalpels Disposable plastic syringes Culture flasks

Move

1. After washing and cutting the tissue (see Steps 1 and 2 of Protocol 12.5), chop the tissue to 3-5 mm³ size and place a few pieces at a time on a stainless steel or polypropylene sieve with a pore size of 1 mm (sieve or a series of sieves with pore sizes of 100 μm to 20 μm, or Falcon's Cell Filter) placed on a 9 cm Picotape Petri dish or in a centrifuge tube.





2. Using the wick of a syringe (disposable plastic syringe, 2 ml or 5 ml), apply gentle pressure to pass the tissue through the mesh into the medium, and aspirate the medium (growth medium, e.g., DMEM/F12 with 10 % fetal bovine serum) and blow it through the mesh to wash the cells down.

3. Aspirate the separated tissue into a 100 μm pore size sieve and repeat step 2.

4. The cell suspension obtained at this point can be diluted for inoculation, or further passed through a 20 μm screen in order to obtain a single cell suspension. In general, the higher the degree of separation of the cell suspension, the higher the shear force and the lower the survival rate.

5. Dilute the cell suspension to ^2×105 /ml, ^1×106 /ml or ^2×106 /ml with culture medium and inoculate into culture flasks.

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