Diffusion chamber culturing of haematopoietic cells: methodological investigations and improvement of the technique.

Mouse bone marrow cells were cultured to determine some basic characteristics of the diffusion chamber (DC) technique. The main findings were as follows: (i) Incorporated 3H-thymidine was conveniently measured after deposition of a cell sample on glass fibre discs followed by methanol washing. Varying the specific activity from approximately 2-approximately 20 Ci/mmole did not affect the relationship between DC cellularity and isotope incorporation. Isotope uptake was similar regardless of whether 3H-thymidine of high or low specific activity had been used. (ii) Higher cell yields were obtained when Millipore or Acropor filters were heat-sealed rather than glued to the plastic rings, when Millipore filters were moistened before DC filling, when we omitted gluing the closing plugs, and when we used an average pore size of 0.22 mum for the DC walls rather than one of 0.10 mum or 0.45 mum. Varying the ring thickness from 2.0-2.5 mm did not impair the cell growth, nor did removal of a softening agent from the plastic rings improve it. (iii) More cells were retrieved from DC carried by young rather than by older mice. Results were not influenced by sex or strain differences between the donor and the host or by the number of implants, whether single or double, within an individual host. (iv) Adding Ficoll to the pronase solution increased the yield of viable CFU-C. (V) Diffusion rate through the DC walls declined with increasing period of culturing, so that i.p. 3H-thymidine is not a flash label for 7-day cultures, for example. The great variability of 3H-thymidine diffusion into i.p. DC was markedly reduced by in vitro exposure to the unopened DC to the isotope. (vi) DC could be incubated in vitro in a medium devoid of protein for at least 6 hours without a fall in 3H-thymidine incorporation rate or CFU-C content, provided that pH was kept constant.